Novel resveratrol-based flavonol derivatives: Synthesis and anti-inflammatory activity in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2019.05.004

In order to discover novel anti-inflammatory agents, total thirty-seven new resveratrol-based flavonol derivatives were designed and synthesized. All compounds have been screened for their anti-inflammatory activity by evaluating their inhibition effect of LPS-induced NO production in RAW 264.7 macrophages. Their toxicity was also assessed in vitro. Structure-activity relationships (SARs)have been concluded, and finally 2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-4H-chromen-4-one was found to be the most active scaffold with low toxicity. This compound could significantly decrease productions of NO, IL-6 and TNF-α with IC₅₀ values of 1.35, 1.12 and 1.92 μM, respectively in RAW 264.7 macrophages. Preliminary mechanism studies indicated that it could inhibit the expression of TLR4 protein, resulting in activation of the NF-?B cell signaling pathway. The in vivo anti-inflammatory activity of this compound could reduce pulmonary inflammation by mouse model of LPS-induced acute lung injury (ALI). We believe these findings would further support studies of rational design of more efficient acute lung injury regulatory inhibitors.

Full text of DOI:10.1016/j.ejmech.2019.05.004

Accepted Manuscript
Novel resveratrol-based flavonol derivatives: Synthesis and anti-inflammatory activity
in vitro and in vivo
Liu Zeng Chen, Li Yao, Ming Ming Jiao, Jing Bo Shi, Yue Tan, Ban Feng Ruan, Xin
Hua Liu
PII:
S0223-5234(19)30407-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.004
EJMECH 11314
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 January 2019
Revised Date: 30 April 2019
Accepted Date: 2 May 2019
Please cite this article as: L.Z. Chen, L. Yao, M.M. Jiao, J.B. Shi, Y. Tan, B.F. Ruan, X.H. Liu, Novel
resveratrol-based flavonol derivatives: Synthesis and anti-inflammatory activity in vitro and in vivo,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.004.
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Graphical abstract
Novel resveratrol-based flavonol derivatives: synthesis and anti-inflammatory
activity in vitro and in vivo
New resveratrol-based flavonol derivatives with anti-inflammatory were
synthesized. Among them, one compound could significantly decreased production
of NO, IL-6 and TNF-α LPS-stimulated. Preliminary mechanism indicated that it
could inhibit expression of TLR4, resulting in activation cell signaling pathway
NF-ĸB and MAPK. The in vivo anti-inflammatory activity was determined by
LPS-induced acute lung injury.

ACCEPTED MANUSCRIPT
Novel resveratrol-based flavonol derivatives: synthesis and anti-inflammatory activity
in vitro and in vivo
Liu Zeng Chen a, Li Yao b, Ming Ming Jiao a Jing Bo Shi a, Yue Tan a, Ban Feng Ruan b, *
and Xin Hua Liu a,*
a School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases
Anhui Medical University, Hefei, 230032, P. R. China
bSchool of Food and Biological Engineering, Hefei University of Technology, Hefei,
230009, P. R. China
Abstract: In order to discover novel anti-inflammatory agents, total thirty-seven new
resveratrol-based flavonol derivatives were designed and synthesized. All compounds have
been screened for their anti-inflammatory activity by evaluating their inhibition effect of
LPS-induced NO production in RAW 264.7 macrophages. Their toxicity was also assessed
in vitro. Structure-activity relationships (SARs) have been concluded, and finally
2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-4H-chromen-4-one was found to
be the most active scaffold with low toxicity. This compound could significantly decrease
productions of NO, IL-6 and TNF-α with IC50 values of 1.35, 1.12 and 1.92 µM,
respectively in RAW 264.7 macrophages. Preliminary mechanism studies indicated that it
could inhibit the expression of TLR4 protein, resulting in activation of the NF-ĸB cell
signaling pathway. The in vivo anti-inflammatory activity of this compound could reduce
pulmonary inflammation by mouse model of LPS-induced acute lung injury (ALI). We
believe these findings would further support studies of rational design of more efficient
acute lung injury regulatory inhibitors.
Keywords: Resveratrol-based flavonol; design; synthesis; anti-inflammatory
_________________
*Corresponding author.
Tel.: +86 551 2901438; fax: +86 551 2901438. E-mail addresses: bf_ruan@163.com (Ban Feng Ruan).
Tel.: +86 551 65172353; fax: +86 551 65161115. E-mail addresses: xhliuhx@163.com (Xin Hua Liu).
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1. Introduction
Pro-inflammatory cytokines play an important role in the defense of disease [1,2].
however, uncontrolled and excess release of pro-inflammatory mediators such as NO,
interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-1β (IL-1β), tumor necrosis factor
alpha (TNF-α) and IL-8 can lead to multiple types of inflammatory diseases such as acute
lung injury (ALI) [3-6]. ALI is a critical illness caused by excessive inflammation, with the
manifestations of acute respiratory distress, non-cardiogenic pulmonary edema and
hypoxemia [7-9]. Many cytokines, such as IL-6, IL-1β and TNF-α have been confirmed to
regulate the pathogenesis of ALI through a series of cell cytokine signaling pathways
[10,11]. Despite airway management and protective ventilation strategies have been
advanced, the mortality rate among ALI patients remains high due to lack of effective
drugs [12,13]. Therefore, discovery of highly effective drugs and therapies inhibiting the
production and release of pro-inflammatory cytokines for treatment of ALI is urgent.
It has been reported that LPS works by activating its major receptor TLR4, triggering
the innate immune systems [14,15], with subsequent activation of nuclear factor (NF-κB)
and mitogen-activated protein kinases (MAPK) to induce the release of pro-inflammatory
cytokines [16]. Upon stimulation with LPS, TLR4 initiates the series of signaling cascades
that result in activation of NF-κB and MAPK to induce the release of pro-inflammatory
cytokines such as NO and IL-6 [17].
Resveratrol, a natural product with stilbene moiety which plays an important
anti-inflammatory role [18-22], is the most famous lead template for the drug design and
discovery [23-25]. In our previous study, based on its good safety profile, diversified
derivatives containing resveratrol were designed and synthesized. Among them,
resveratrol-based cinnamic ester hybrids indicated good anti-inflammatory activity through
reducing proinflammatory cytokines due to its inhibitory effect on the NF-κB signaling
pathway. Unfortunately, the activity of these compounds in vivo is not satisfactory. We
focused on this structure and speculated that the α, β unsaturated ketone moiety was prone
to Michael addition reaction in vivo, which should be affect activity (Figure 1). Based on
this, we intend to retain the resveratrol skeleton fragments while forming a rigid plane of α,
β unsaturated ketone, and look forward to improving activity. So, as part of our continuous
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interest in search of novel resveratrol analogs as anti-inflammatory agents with good
bioavailability, other active unit was considered to introduce. As is known, flavonoids in
citrus have been widely studied for their potential anti-inflammatory actions, subclasses of
dietary flavonoids can alter both bioavailability and bioactivity [26-28]. Therefore, based
on these two naturally and pharmaceutically active scaffolds, some new resveratrol-based
flavonoid were synthesized (Figure 1), in-house compounds library screening showed that
most compounds had no toxicity against tumor cell lines. Further activity tests showed that
the toxicity of RAW 264.7 cells was also low. We then evaluated their anti-inflammatory
activity in vitro and in vivo.
Figure 1
2. Results and discussion
2.1 Chemistry
The synthesis of resveratrol aldehyde derivatives (3a~3d) fragment was exhibited in
Scheme 1. Compounds (2a~2d) were obtained by reacting pterostilbene with a brominated
alkane in the presence of potassium carbonate (K2CO3) and tetrabutylammonium bromide
(TBAB) in acetone under reflux condition. With the Vilsmeier reaction of compounds
2a~2d using POCl3 and DMF afforded the key aldehyde intermediates 3a~3d.
Resveratrol-based flavone derivatives 5a~5v were outlined in Scheme 2. Reaction of
resveratrol aldehyde derivatives (3a~3d) with hydroxyacetophenone derivatives in ethanol
in the presence of pyrolidine resulted to compounds 4a~4v. Then, resveratrol chalcone
derivatives were treated with iodine (I2) in DMSO under reflux, compounds 5a~5v were
obtained, their structures were shown in Table 1. Resveratrol flavonol derivatives (7a~7o)
were prepared following the procedure described by Scheme 3. The synthesis method of
compounds 6a~6o is the same as compounds 4a~4v. Then, resveratrol flavonol derivatives
(7a~7o) were prepared from compounds 6a~6o by treatment with NaOH and 30% H2O2 in
the presence of methanol at 40 °C. The structures of 7a~7o shown in Table 2. All
compounds were purified by recrystallization or column chromatography and characterized
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by 1H NMR, 13C NMR and HRMS (ESI).
Schemes 1~3
Tables 1~2
2.2 Crystal structure of compounds 5r and 5u.
The structure of compound 5r was determined by X-ray crystallography (Figure 2).
Crystallographical data: C30H30O6, Triclinic, space group p-1; a = 9.8786 (7), b =
11.6966(11), c = 22.6393 (17) (Å);
α = 93.908 (7), β = 98.968 (6), γ = 94.751 (6) (°), V =
2566.3 (4) nm3, T =293 (2) K, Z = 4, Dc = 1.259 g/cm3, F(000) = 1032, Reflections
collected/unique =17725/10093, Data/restraints/parameters = 10093/0/657, Goodness of fit
on F2 = 1.073, Fine, R1 = 0.1167, wR(F2) = 0.1589. Compound 5u: C33H28O6, Triclinic,
space group p-1; a = 10.1966 (13), b = 16.739 (3), c = 17.174(3) (Å);
α = 65.938 (16), β =
76.808 (14),
γ
= 88.373(12) (°), V = 2599.0(7) nm3, T = 293(2) K, Z = 4, Dc = 1.33 g/cm3,
F(000) = 1096, Reflections collected/unique = 17809/10212, Data/restraints/parameters =
10212/0/709, Goodness of fit on F2 = 1.025, Fine, R1 = 0.1646, wR(F2) = 0.23.
Crystallographic data (excluding structure factors) for the structures have been deposited
into the Cambridge Crystallographic Data Center.
(Figure 2)
2.3 Inhibitory activities against LPS-induced NO release
NO is an important pro-inflammatory mediator [29]. Excessive production of NO was
found play an important role in many inflammatory diseases [30]. So NO inhibitors
accepted that may offer potential opportunity to cure inflammatory diseases [31,32]. RAW
264.7 cells as useful cell model in screening anti-inflammatory drugs, since their
stimulation by LPS induces and secretion of pro-inflammatory cytokines such as NO, IL-6
and TNF-α. In order to evaluate the anti-inflammatory activity of synthetic compounds,
RAW 264.7 cells were pre-incubated with all compounds (10 µM) for 1 h and treated with
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LPS (0.5 µg/mL ) for 24 h. The cell conditioned medium was collected, the NO in the
media was detected by Griess Reagent assay. The screening results indicated that most of
the synthetic compounds could reduce the LPS-induced NO secretion at 10 µM (Figure 3).
Among them, compounds 5e and 5h showed moderate inhibition of NO compared to that
of the positive control Celecoxib. It is noteworthy that compounds 7d, 7f, 7i, 7k and 7m
exhibited stronger inhibition of NO production compared with the referece compounds
celecoxib and resveratrol. According to this, SARs can be easily concluded that
introduction of the hydroxyl group to flavonoid is beneficial to NO release activity.
Figure 3
2.4 Assessment of toxicity
Preliminary screening results showed that some compounds had good
anti-inflammatory activity. To confirm the need for further evaluation, we then evaluated
the cytotoxicity of selected compounds using the MTT assay on the RAW264.7 cell. As
showned in Figure 4, most of the compounds shown low toxicity at 20 µM. Compounds 5f,
5o, 5u, 5v, 7b and 7n show weak cytotoxicity. Therefore, these compounds are valuable
for further evaluation.
Figure 4
2.5 Inhibition of cytokine production by the active compounds
Two important cytokines, IL-6 and TNF-α have been shown to exert regulating effects
on the pathogenesis of ALI through a series of cytokine signaling pathways [33,34]. So,
compounds with good NO activity were further tested for inhibition of others inflammatory
factors as IL-6 and TNF-α. Cells were pretreated with different concentrations of
compounds. The results indicated that most compounds had good IL-6 and TNF-α activity.
Among them, compounds 7d, 7f and 7i were selected for further assessment of their
concentration dependent inhibitory effects against LPS-induced NO, TNF-α, IL-6 release.
Macrophages were pretreated with compounds 7d, 7f and 7i, in a series of concentrations
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(10, 5, 2.5, 1.25 and 0.625 µM) for 1 h and with LPS (0.5 µg/ mL) for 24 h. As shown in
Figure 5, these compounds significantly decreased NO (IC50s 1.78, 1.35 and 4.83 µM,
respectively), IL-6 (IC50s 2.37, 1.12 and 5.06 µM, respectively) and TNF-α (IC50s 5.00,
1.92 and 7.34 µM, respectively) secretion in a concentration-dependent manner. Based on
above, compound 7f is the most prominent one. Thus, compound 7f was used as the title
compound for next mechanism exploration.
Figure 5
2.6 Mechanism explorations of anti-inflammatory activity
2.6.1 Suppression of LPS-induced inflammatory response
NO, being an important signaling molecule, which is also importantly related to
modulation expression of iNOS and COX-2 [35]. Activation of cellular pathways leads to
high expression of iNOS and COX-2 protein. Thus, inhibitory effects of compound 7f on
LPS-mediated expressions of iNOS and COX-2 were analyzed by Western blot. As shown
in Figure 6, the LPS (0.5 µg/mL) stimulation for 24 h could be markedly augmented iNOS
and COX-2 expression. However, compound 7f concentration dependently suppressed
LPS-induced iNOS and COX-2 expression. This results once more demonstrated that
compound 7f prevented LPS-induced inflammatory response in macrophages.
Figure 6
2.6.2 Inhibition of LPS-induced ERK and P38 signaling activation
Protein TLR4 is a key protein of the LPS-activated cellular signaling pathway and has
been reported as critical for the inflammatory response to LPS [30]. When LPS stimulate
cell, TLR4 initiates series of signaling cascades that result in the activation cell signaling
pathway including NF-ĸB and MAPK to induce expression pro-inflammatory proteins
[14,31]. Recent studies found that inhibition of TLR4 expression could decrease the
expressions of NO, IL-6, TNF-α and IL-1β. So we investigated whether compound 7f
inhibited the expression of TLR4 by Western blot. The results confirmed that TLR4
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expression was up-regulated in LPS-induced RAW264.7 cells, which was reversed in a
concentration-dependent manner by pretreated with compound 7f.
NF-ĸB and MAPKs are also known to participate in regulating the inflammatory
process. MAPK, including ERK, p38, and JNK were quite significant in the regulation of
inflammation [35,36]. LPS-stimulated macrophages can activate MAPKs signaling path,
allowing the transcription factors AP-1 translocate into the nucleus and bind to target
promoters turn on transcriptions of inflammatory genes including production of NO,
TNF-α, IL-6 and other inflammatory mediators [30]. Therefore, we detected the effects of
compound 7f on LPS-mediated MAPK signaling activation by Western blot. Consistent
with previous reports of LPS time dependently activation MAPK (JNK, p38 and ERK) and
caused a peak level of phospho-rylated MAPK (JNK2, p38 and ERK) at 15 or 30 min in
RAW264.7 cells. Interestingly, compound 7f only concentration-dependent blocked
LPS-induced ERK phosphorylation and p38 phosphorylation, but not JNK phosphorylation.
Moreover, total protein levels of ERK, JNK and P38 were not affected by LPS and
treatment of compound 7f (Figure 7).
Figure 7
2.6.3 Inhibition of activation of LPS-induced NF-kB signaling pathway
NF-κB is one of the principal factors for the production proinflammatory cytokines
associated with LPS-induced signaling pathways [36]. Among NF-κB family, the
transcription factor P65 plays the most important role in the development of inflammation
[37-38]. A large number of inflammation cytokines stimuli such as LPS, TNF-α and IL-1β
can activation of IκB kinase (IKK), which in turn phosphorylates IκB. Phosphorylates IκB
results in its ubiquitination and degradation by the proteasome, allowing the liberated
NF-κB to translocate into the nucleus and bind to target promoters turn on transcriptions of
inflammatory genes. Herein, we analyzed them by Western blot. The results showed
compound 7f could be effect of IκB proteins phosphorylation and degradation (Figure 8).
Meanwhile, it maybe inhibits NF-κB p65 translocate into the nucleus.
Figure 8
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2.7 Molecular docking
It has been reported that Tak1 is traditionally accepted as the primary LPS receptor and
critical for the inflammatory response to LPS [14-17]. Upon stimulation with LPS, TLR4
initiates series of signaling cascades that result in activation of TAK1 signal pathway. Tak1
could activate NF-κB and MAPK, then induce the release of pro-inflammatory cytokines.
In order to elucidate the mechanism by which compound 7f can induce the release of
pro-inflammatory cytokine. Molecular docking of this compound into binding site of
human TAK1 was performed (Discovery Studio 2018). The result shows that compound 7f
may well bind to TAK1 and can form interaction with Val42, Val50, Cys174, Leu163. Its
4-position carbonyl forms a stable hydrogen bond interaction with Tyr106. These results
suggested that the anti-inflammatory activity of compound 7f might be bind to TAK1
protein (Figure 9).
Figure 9
2.8 In vivo activity of compound 7f
To evaluate the activity in vivo, compound 7f was next evaluated with the ALI rat
model. C57/BL6 mice were treated by intraperitoneal injection with compound 7f (15
mg/kg), and after 30 min, were challenged with 5 mg/kg LPS by intratracheal injection.
Myeloperoxidase (MPO) is released from the cytoplasmic granules of activated phagocytes,
and as such, measurement of MPO activity in whole lung homogenates reflects the
accumulation of neutrophils in the lungs. As showned in Figure 10A, LPS significantly
increased MPO activity of lung tissue compared to the control group, in which the
administration of compound 7f effectively prevented the increase. Besides, due to LPS
challenge significantly reduce body weight, however, compound 7f seemed to have little
effect on the weight loss induced by LPS. Lung wet/dry weight ratio was calculated to
assess pulmonary edema. LPS significantly increased the lung Wet/Dry ratio (W/D).
However, compound 7f was able to reduce or prevent lung injury in ALI mice (Figure
10B). In addition, to further research the protective effect of compound 7f against
LPS-induced ALI. As depicted in Figure 10C, with the challenge of LPS, the survival rate
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of ALI mice at 24 h was 60% while administration of compound 7f improved the survival
rate of 90%. When LPS challenge was extended to 48 h, most mice dead. Compound 7f
(20 mg/kg) could markedly increase the survival rate of ALI mice from 10% to 50%
(Figure 10D). To assess histological changes in LPS-challenged mice following treatment
with compound 7f, we performed hematoxylin and eosin (H&E) staining (Figure 10E).
LPS instillation led to significant pro-inflammatory alterations, alveolar hemorrhage,
including lung edema, inflammatory cell infiltration, and destruction of alveolar structure.
These histopathological changes were improved with treatment of either 10 and 20 mg/kg
of compound 7f. These studies showed that compound 7f had a protective effect on
LPS-induced histopathological changes in a mouse model of ALI.
In order to determine what is the toxicity profile of the most active compound 7f on
experimental animals, mice were treated with compound 7f (20 mg/kg) solutions by
intraperitoneal injection. The mice live normally, no differences compared to the control
group. lung sections were subjected to hematoxylin and eosin staining. we couldn’t find
significant pro-inflammatory alterations, alveolar hemorrhage, including lung edema,
inflammatory cell infiltration, and destruction of alveolar structure. Lung tissues showed a
normal structure and no histopathological change under a light microscope (Figure 11).
Figures 10~11
3. Conclusions
In summary, based on finding novel compounds with activity of acute lung injury,
thirty-nine resveratrol-based flavonol derivatives were designed and synthesized. The
initial evaluation results showed that most compounds had good NO inhibitory activity and
low toxicity. According to SARs, the introduction of a hydroxyl group into flavonoid is
beneficial to anti-inflammatory activity. Specifically, the title compound 7f could inhibit
IL-6, NO and TNF-α secretion in a dose-dependent manner. The preliminary mechanism
indicated that this compound suppressed LPS-induced expressions of iNOS and COX-2,
and productions of IL-6, TNF-α and NO through NF-κB/MAPK signaling pathway in a
concentration dependent manner (Figure 12). The further study in vivo showed that the title
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compound 7f effectively reduced LPS-induced pulmonary inflammation and acute lung
injury in mouse model. Compared to resveratrol-based cinnamic ester hybrids (our
previous work) and the title compounds synthesized in this resveratrol-based flavonols
(this work), the anti-inflammatory activity of the title compound was dramatically
enhanced at the cellular level, and the activity of acute lung injury was further confirmed
by animal experiments in vivo. In addition, the toxicity of these compounds is lower than
previous compounds [22].
Figure 12
4. Experimental section
4.1 Chemistry
In general, all reagents used in the synthesis were obtained from Aladdin. Adamas,
Tan soole et al. without further purifications. Reactions were monitored by analytical
thin-layer chromatography (TLC) and visualized under UV light (λ=254 or 365 nm).
Purification by chromatography column were carried on using silica gel (200-300 meshes).
Fourier transform mid-infrared spectra were measured using a Nicolet 6700 spectrometer
(Thermo Fisher Scientific Inc., Madison, WI) with SMART iTR attenuated total
1
reflectance (ATR) accessory. All the H and 13C NMR (Nuclear Magnetic Resonance)
spectra were recorded either with a Agilent 400 or 600 MHz spectrometer. The ESI-MS
spectra were recorded on a Mariner System 5304 Mass spectrometer. Melting points were
determined on a XT4MP apparatus (Taike Corp., Beijing, China), and uncorrected.
4.2 Crystallographic studies
Compounds 5r and 5u were chosen for X-ray diffraction analysis performed on a
BRUCKER SMART APEX-CCD diffractometer equipped with a graphite monochromatic
MoKa radiation (λ = 0.71073 A) radiation at 293(2) K. A total reflections were collected in
the range of 0.97<θ<26.1o by using a ψ-ω scan mode with independent ones, of which
I>2σ(I) were observed and used in the succeeding refinements. The data set were corrected
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by SADABS program and the structure were solved by direct methods with SHELXS-97
and refined by full-matrix least-squares method on F2 with SHELXL-97 [39]. The
non-hydrogen atoms were refined anisotropically, and the hydrogen atoms were added
according to theoretical models. The structures were refined by full-matrix least-squares
method on F2 with SHELXT-97.
4.3 Synthesis of resveratrol-based flavone derivatives 5a~5v
A solution containing of compounds 4a~4v (1 mmol, 1.0 equiv) and I2 (0.01 mmol,
0.01 equiv) in dimethyl sulfoxide (15 mL) was stirred for 4-6 h at 130 °C. Then the
resulting mixture was slowly added to a solution of ice-water, stirred for 2 h and filtered to
obtain the crude product. Which was purified by column chromatography on silica gel
(EtOAc/petroleum ether = 1:3), to afford the corresponding pure products 5a~5v (Scheme
1). The final products provided the following data.
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-4H-chromen-4-one (5a). White solid,
Yield: 90%, Mp: 193-197 °C. 1H NMR (600MHz, CDCl3) δ 8.27 (d, J = 7.8 Hz, 1H), 7.65
(t, J = 7.7 Hz, 1H), 7.43 (dd, J = 14.9, 7.8 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H), 7.07-6.98 (m,
1H), 6.92-6.83 (m, 2H), 6.80 (d, J = 8.6 Hz, 2H), 6.44 (d, J = 21.6 Hz, 2H), 3.92 (s, 3H),
3.78 (t, J = 5.8 Hz, 6H). 13C NMR (151 MHz, CDCl3): δ 173.36 (s), 163.97 (s), 162.31 (s),
160.96 (s), 157.47 (s), 146.85 (s), 142.56 (s), 141.89 (s), 135.75 (s), 133.61 (s), 132.11 (s),
130.51 (s), 127.14 (s), 127.04 (s), 124.85 (s), 128.08 (s), 120.15 (s), 115.78 (s), 113.98 (s),
103.94 (s), 100.63 (s), 57.61 (s), 57.46 (s), 56.89 (s). MS (ESI): 415.1440. MS(ESI):
415.1440 (C26H23O5, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-7-methyl-4H-chromen-4-one (5b). White
1
solid, Yield: 61%, Mp: 186-189 °C. H NMR (600 MHz, DMSO) δ 7.95 (d, J = 8.1 Hz,
1H), 7.41 (s, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.31 (d, J = 8.1 Hz,1H), 7.25 (d, J = 16.1 Hz,
1H), 7.01 (t, J = 4.2 Hz, 1H), 6.87-6.83 (m, 3H), 6.62 (d, J =1.9 Hz, 1H), 6.23 (s, 1H), 3.88
(s, 3H), 3.75 (s, 3H), 3.69 (d, J = 3.7 Hz, 3H), 2.42 (s,3H). 13C NMR (101 MHz, CDCl3): δ
175.27 (s), 162.78 (s), 160.29 (s), 159.73 (s),156.29 (s), 147.14 (s), 145.65 (s), 142.22 (s),
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139.47 (s), 131.87 (s), 130.13 (s), 128.89(s), 126.93 (s), 124.89(s), 123.64 (s), 120.73 (s),
118.87 (s), 99.87(s), 98.79 (s), 113.18(s), 110.76 (s), 56.85 (s), 55.81 (s), 55.95 (s), 20.95
(s). MS(ESI): 429.1697 (C27H25O5, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-6-methyl-4H-chromen-4-one (5c). White
solid, Yield: 59%, Mp: 187-192 °C. 1H NMR (600 MHz, CDCl3) δ 8.07(s, 1H), 7.46 (d, J =
8.0 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 16.0 Hz, 1H),
6.92-6.84 (m, 2H), 6.80 (d, J = 8.5 Hz, 2H), 6.44 (d, J = 29.6 Hz, 2H), 3.92 (s, 3H), 3.78 (d,
J = 1.7 Hz, 6H), 2.48 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 168.94(s), 161.46 (s),
158.79 (s), 156.89 (s), 154.56 (s), 143.94 (s), 136.73 (s), 135.54 (s), 134.79 (s), 134.12 (s),
130.98 (s), 128.93 (s), 128.14 (s), 125.17 (s), 122.81 (s), 120.91 (s), 117.94 (s), 113.89 (s),
110.95 (s), 100.96 (s), 98.80 (s), 58.03 (s), 54.73 (s), 52.43 (s), 22.98 (s). MS(ESI):
429.1694 (C27H25O5, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-6,7-dimethyl-4H-chromen-4-one
(5d).
White solid, Yield: 72%, Mp: 203-207 °C. IR (ATR Diamond Crystal, cm-1): v 2972, 1620,
1596, 1461, 1332, 1302, 1159, 1177, 967, 830; 1H NMR (600 MHz, CDCl3) δ 7.98 (d, J =
35.8 Hz, 1H), 7.28 (d, J = 8.5 Hz, 2H), 7.22 (s, 1H), 7.03 (d, J =16.0 Hz, 1H), 6.91-6.84 (m,
2H), 6.80 (d, J = 8.5 Hz, 2H), 6.46 (s, 1H), 6.38 (s, 1H), 3.92 (s, 3H), 3.77 (s, 6H), 2.37 (d,
J = 9.2 Hz, 5H). 13C NMR (101 MHz, CDCl3): δ 179.68 (s), 163.59 (s), 158.97 (s), 157.81
(s), 156.16 (s), 145.27 (s), 145.00 (s), 138.98 (s), 137.50 (s), 135.64 (s), 130.29 (s), 128.97
(s), 128.15 (s), 123.83 (s), 122.66 (s), 109.98 (s), 107.67 (s), 106.88 (s), 103.62 (s), 101.30
(s), 99.00 (s), 57.02 (s), 56.54 (s), 56.19 (s), 21.65 (s), 20.19 (s). MS(ESI): 443.1854
(C28H27O5, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-6-methoxy-4H-chromen-4-one
(5e).
Yellow solid, Yield: 66%, Mp: 168-170 °C. 1H NMR(600 MHz, CDCl3) δ 7.64 (d, J = 2.9
Hz, 1H), 7.39 (d, J = 9.1 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 7.26-7.24 (m, 1H), 7.03 (d, J =
16.0 Hz, 1H), 6.87 (d, J = 15.9 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 6.44 (d, J = 22.9 Hz, 2H),
13
3.92 (s, 6H), 3.78 (s, 6H). C NMR (101 MHz, DMSO): δ 178.09 (s), 164.72 (s), 161.29
12

ACCEPTED MANUSCRIPT
(s), 159.73 (s), 156.44 (s), 149.82 (s), 146.91 (s), 141.76 (s), 140.53 (s), 131.79 (s), 129.02
(s), 125.83 (s), 122.48 (s), 120.70 (s), 117.63 (s), 115.05 (s), 114.28 (s), 112.05 (s), 100.96
(s), 98.92 (s), 96.80 (s), 57.86 (s), 55.09 (s), 53.51 (s), 45.05 (s). MS(ESI): 445.1650
(C27H25O6, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-6,7-dimethoxy-4H-chromen-4-one (5f).
1
Yellow solid, Yield: 42%, Mp: 188-192 °C. H NMR (600 MHz, DMSO) δ 7.39 (s, 1H),
7.33 (d, J = 8.7 Hz, 2H), 7.25 (d, J = 16.2 Hz, 1H), 7.20 (s, 1H), 7.00 (d, J = 1.8 Hz, 1H),
6.85 (t, J = 11.6 Hz, 2H), 6.81 (d, J = 16.2 Hz, 1H), 6.61 (t, J = 3.0 Hz, 1H), 6.19 (s, 1H),
13
3.88 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.75 (s, 3H), 3.70 (s, 3H). C NMR (101 MHz,
DMSO): δ 178.09 (s), 164.72 (s), 161.29 (s), 159.73 (s), 156.44 (s), 149.82 (s), 146.91 (s),
141.76 (s), 140.53 (s), 131.79 (s), 129.02(s), 125.83 (s), 122.48 (s), 120.70 (s), 117.63 (s),
115.05 (s), 114.28 (s), 112.05 (s), 100.96 (s), 98.92 (s), 96.80 (s), 57.86 (s), 55.09 (s), 53.51
(s), 42.05(s), 39.67 (s). MS(ESI): 475.1760 (C28H27O7, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-7-fluoro-4H-chromen-4-one (5g). Yellow
1
solid, Yield: 78%, Mp: 186-191 °C. H NMR (600 MHz, CDCl3) δ 8.28 (dd, J = 8.5, 6.5
Hz, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.14 (dd, J = 20.3, 8.8 Hz, 2H), 7.03 (d, J = 16.0 Hz,
1H), 6.89-6.78 (m, 4H), 6.49-6.39 (m, 2H), 3.93 (s, 3H), 3.79 (d, J = 1.1 Hz, 6H). 13C
NMR (101 MHz, DMSO): δ 185.91 (s), 174.63 (s), 165.87 (s), 155.84 (s), 150.16 (s),
147.85 (s), 145.32 (s), 139.04 (s), 137.53 (s), 129.72 (s), 127.91 (s), 125.95 (s), 123.96 (s),
123.56 (s), 120.94 (s), 118.90 (s), 115.64 (s), 113.64 (s), 108.12 (s), 102.95 (s), 98.70 (s),
53.88 (s), 50.52 (s), 45.65 (s). MS(ESI): 433.1449 (C26H22FO5, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-6-fluoro-4H-chromen-4-one (5h). White
solid, Yield: 59%, Mp: 216-220 °C. IR (ATR Diamond Crystal, cm-1): v 3002, 2936, 2836,
1640, 1568, 1474, 1340, 1240, 1085, 843; 1H NMR (600 MHz, CDCl3) δ 7.91 (dd, J = 8.1,
2.4 Hz, 1H), 7.45 (dd, J = 9.0, 3.9 Hz, 1H), 7.38 (dd, J = 11.4, 5.1 Hz, 1H), 7.28 (d, J = 8.5
Hz, 2H), 7.03 (d, J = 16.0 Hz, 1H), 6.91-6.79 (m, 4H), 6.45 (d, J = 25.2 Hz, 2H), 3.93 (s,
3H), 3.79 (d, J = 2.5 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 171.91 (s), 161.93 (s),
13

ACCEPTED MANUSCRIPT
159.20 (s), 158.84 (s), 157.16 (s), 151.85 (s), 146.32 (s), 140.04 (s), 138.63 (s), 130.92 (s),
129.21 (s), 127.95 (s), 123.16 (s), 122.56 (s), 121.74 (s), 121.20 (s), 114.14 (s), 111.44 (s),
109.12 (s), 100.95 (s), 97.78 (s), 55.88 (s), 55.52 (s), 55.05 (s). MS(ESI): 433.1441
(C26H22FO5, [M+H]+).
(E)-8-chloro-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-4H-chromen-4-one (5i). White
solid, Yield: 58%, Mp: 231-234 °C. 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 7.9 Hz, 1H),
7.66 (t, J = 7.7 Hz, 1H), 7.47-7.40 (m, 2H), 7.31-7.24 (m, 2H), 6.88 (dd, J = 14.6, 8.7 Hz,
2H), 6.81 (d, J = 8.4 Hz, 2H), 6.47 (s, 2H), 3.93 (s, 3H), 3.78 (d, J = 4.7 Hz, 6H). 13C NMR
(101 MHz, DMSO): δ 182.91 (s), 167.63 (s), 160.16 (s), 159.864 (s), 158.06 (s), 152.75 (s),
148.02 (s), 141.24 (s), 139.53 (s), 131.52 (s), 128.91 (s), 128.05 (s), 122.16 (s), 121.56 (s),
120.74 (s), 120.20 (s), 115.14 (s), 112.14 (s), 108.62 (s), 100.35 (s), 96.78 (s), 57.12 (s),
54.82 (s), 52.05 (s). MS(ESI): 449.1147 (C26H22ClO5, [M+Na]+).
(E)-7-chloro-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-4H-chromen-4-one (5j). White
solid, Yield: 68%, Mp: 221-225 °C. 1H NMR(400 MHz, CDCl3) δ 8.17 (d, J = 8.4 Hz, 1H),
7.29 (d, J = 8.5 Hz, 2H), 7.25 (s, 1H), 7.04 (d, J = 16.1 Hz, 1H), 6.89 (d, J = 18.3 Hz, 2H),
13
6.81 (d, J = 8.4 Hz, 2H), 6.46 (s, 2H), 3.93 (s, 3H), 3.79 (s, 6H). C NMR (101 MHz,
CDCl3): δ 187.51 (s), 175.63 (s), 168.26 (s), 159.84 (s), 158.16 (s), 156.15 (s), 149.12 (s),
145.14 (s), 140.43 (s), 133.62 (s), 129.91 (s), 128.25 (s), 122.46 (s), 120.86 (s), 119.74 (s),
118.20 (s), 115.64 (s), 112.04 (s), 109.62 (s), 99.35 (s), 95.78 (s), 58.12 (s), 52.82 (s), 50.05
(s). MS(ESI): 471.0970 (C26H22ClO5 , [M+Na]+).
(E)-6-chloro-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-4H-chromen-4-one (5k). White
solid, Yield: 71%, Mp: 227-229 °C. 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.31-7.25
(m, 2H), 7.03 (d, J = 16.1 Hz, 1H), 6.88 (d, J = 16.8 Hz, 2H), 6.80 (d, J = 8.5 Hz, 2H), 6.45
(d, J = 7.3 Hz, 1H), 6.39 (s, 1H), 3.92 (s, 3H), 3.78 (s, 6H). 13C NMR (101 MHz, CDCl3): δ
179.51 (s), 170.73 (s), 168.66 (s), 160.24 (s), 158.56 (s), 154.65 (s), 148.12 (s), 146.34 (s),
137.73 (s), 134.62 (s), 128.93 (s), 127.85 (s), 124.46 (s), 119.86 (s), 113.84 (s), 110.78 (s),
108.64 (s), 100.04 (s), 98.62 (s), 78.35 (s), 74.78 (s), 46.88 (s), 45.82 (s), 41.65 (s).
MS(ESI): 471.0970 (C26H21ClO5, [M+Na]+).
14

ACCEPTED MANUSCRIPT
(E)-7-bromo-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-4H-chromen-4-one (5l). White
solid, Yield: 75%, Mp: 219-220 °C. IR (ATR Diamond Crystal, cm-1): v 2988, 2900, 1647,
1595, 1511,1418, 1333, 1080, 963, 829; 1H NMR (600 MHz, CDCl3) δ 8.13 (d, J = 8.5 Hz,
1H), 7.64 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 16.0 Hz,
13
1H), 6.87-6.80 (m, 3H), 6.43 (d, J = 31.4 Hz, 2H), 3.93 (s, 3H), 3.79 (s, 6H). C NMR
(101 MHz, CDCl3): δ 181.51 (s), 172.87 (s), 169.16 (s), 161.34 (s), 159.46 (s), 154.35 (s),
150.32 (s), 147.64 (s), 134.73 (s), 130.12 (s), 128.23 (s), 126.55 (s), 124.36 (s), 118.86 (s),
113.54 (s), 111.78 (s), 109.74 (s), 101.02 (s), 99.32 (s), 86.35 (s), 79.28 (s), 56.88 (s), 55.12
(s), 40.25 (s). MS(ESI): 515.0465 (C26H21BrO5Na, [M+Na]+).
(E)-6-bromo-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-4H-chromen-4-one (5m). White
1
solid, Yield: 88%, Mp: 225-229 °C. H NMR (600 MHz, DMSO) δ 8.14 (d, J = 2.1 Hz,
1H), 7.93 (dd, J = 8.9, 2.2 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.39 (d, J = 8.6 Hz, 2H), 7.25
(d, J = 16.1 Hz, 1H), 7.01 (s, 1H), 6.91-6.86 (m, 1H), 6.85 (d, J = 8.6 Hz, 2H), 6.62 (s, 1H),
6.34 (s, 1H), 3.89 (s, 3H), 3.75 (s, 3H), 3.70 (s, 3H). 13C NMR (101 MHz, CDCl3): δ
180.51 (s), 176.87 (s), 162.96 (s), 160.34 (s), 158.96 (s), 154.15 (s), 151.32 (s), 148.34 (s),
136.73 (s), 131.12 (s), 129.53 (s), 126.65 (s), 125.16 (s), 119.86 (s), 113.44 (s), 110.78 (s),
106.74 (s), 101.32 (s), 98.32 (s), 85.35 (s), 77.28 (s), 55.84 (s), 53.42 (s), 50.25 (s).
MS(ESI): 515.0465 (C26H21BrO5Na, [M+Na]+).
(E)-2-(2-(4-ethoxystyryl)-4,6-dimethoxyphenyl)-4H-chromen-4-one (5n). White solid,
Yield: 93%, Mp: 178-180 °C. IR (ATR Diamond Crystal, cm-1): v 2981, 2901, 1636, 1600,
1
1566, 1461, 1238, 1178, 1051, 755; H NMR (400 MHz, DMSO) δ 8.11 (d, J = 7.1 Hz,
1H), 7.81 (t, J = 7.7 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.37 (d, J =
8.6 Hz, 2H), 7.27 (d, J = 16.1 Hz, 1H), 7.03 (d, J = 1.5 Hz, 1H), 6.87 (dd, J = 19.3, 12.4 Hz,
3H), 6.66 (s, 1H), 6.31 (s, 1H), 3.99 (q, J = 6.9 Hz, 2H), 3.92 (s, 3H), 3.78 (s, 3H), 1.29 (t,
J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 181.53 (s), 174.57 (s), 163.46 (s), 161.24
(s), 159.74 (s), 153.45 (s), 150.46(s), 149.20 (s), 137.93 (s), 130.56 (s), 128.78 (s), 125.93
15

ACCEPTED MANUSCRIPT
(s), 123.59 (s), 118.05 (s), 111.97 (s), 109.64 (s), 106.89 (s), 100.98 (s), 96.83 (s), 84.96 (s),
74.95 (s), 54.43 (s), 52.34 (s), 50.29 (s), 42.89(s). MS(ESI): 451.1516 (C27H24O5Na,
[M+Na]+).
(E)-2-(2-(4-ethoxystyryl)-4,6-dimethoxyphenyl)-6-methoxy-4H-chromen-4-one (5o). White
solid, Yield: 83%, Mp: 167-169 °C. IR (ATR Diamond Crystal, cm-1): v 2971, 2901, 1639,
1
1592, 1568, 1486, 1331, 1156, 1074, 1037, 829, 816; H NMR (600 MHz, CDCl3): δ
8.03(d, J = 3.1 Hz, 1H), 7.86 (s, 1H), 7.55 (s, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.18 (s, 2H),
6.80 (d, J = 16.1 Hz, 1H), 6.61 (s, 2H), 6.51 (d, J = 16.1 Hz, 1H), 6.34 (s, 1H), 6.21 (s, 1H),
13
3.53 (d, J = 3.2 Hz, 6H), 3.23 (s, 3H), 3.01 (dd, 2H), 1.58 (t, J =3.2 Hz, 3H). C NMR
(101 MHz, CDCl3): δ 180.24 (s), 175.23 (s), 165.85 (s), 161.97 (s), 149.74 (s), 143.45 (s),
139.56(s), 128.97 (s), 126.43 (s), 124.56 (s), 120.78 (s), 118.93 (s), 113.59 (s), 107.05 (s),
102.97 (s), 100.64 (s), 98.89 (s), 86.98 (s), 84.83 (s), 80.96 (s), 67.95 (s), 45.43 (s), 42.34
(s), 40.89 (s), 40.09(s), 35.27(s). MS(ESI): 481.1622 (C28H26O6Na, [M+Na]+).
(E)-6-bromo-2-(2-(4-ethoxystyryl)-4,6-dimethoxyphenyl)-4H-chromen-4-one (5p). White
solid, Yield: 88%, Mp: 243-244 °C. IR (ATR Diamond Crystal, cm-1): v 3017, 2978, 2931,
1
2882, 1633, 1568, 1428, 1237, 1085, 803; H NMR (400 MHz, DMSO) δ 8.17 (d, J = 2.5
Hz, 1H), 7.96 (dd, J = 8.9, 2.5 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.40 (d, J = 8.7 Hz, 2H),
7.27 (d, J = 16.1 Hz, 1H), 7.03 (d, J = 2.0 Hz, 1H), 6.94-6.87 (m, 1H), 6.85 (d, J = 8.7 Hz,
2H), 6.65 (d, J = 2.0 Hz, 1H), 6.36 (s, 1H), 3.99 (q, J = 7.0 Hz, 2H), 3.91 (s, 3H), 3.78 (s,
3H), 1.29 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 179.64 (s), 175.13 (s), 163.45
(s), 162.97 (s), 152.85 (s), 150.45 (s), 149.43(s), 126.34 (s), 125.83 (s), 123.78 (s), 121.53
(s), 119.53 (s), 115.73 (s), 108.25 (s), 103.52 (s), 101.47 (s), 100.89 (s), 98.25 (s), 91.33 (s),
83.32 (s), 75.95 (s), 52.55 (s), 49.43 (s), 47.16 (s), 39.96(s). MS(ESI): 529.0621
(C27H23BrO5Na, [M+Na]+).
(E)-2-(2-(4-butoxystyryl)-4,6-dimethoxyphenyl)-4H-chromen-4-one (5q). Yellow solid,
Yield: 89%, Mp: 167-169 °C. IR (ATR Diamond Crystal, cm-1): v 2958, 2931, 2871, 1640,
1596, 1574, 1463, 1350, 1248, 1203, 1156, 1075, 829, 748; 1H NMR (600 MHz, DMSO) δ
16

ACCEPTED MANUSCRIPT
8.08 (dd, J = 7.9, 1.2 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.50 (t, J =
7.5 Hz, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 16.1 Hz, 1H), 7.01 (d, J = 1.7 Hz, 1H),
6.89- 6.84 (m, 1H), 6.83 (d, J = 8.5 Hz, 2H), 6.62 (d, J = 1.6 Hz, 1H), 6.28 (s, 1H), 3.92-
3.89 (m, 2H), 3.89 (s, 3H), 3.75 (s, 3H), 1.65-1.59 (m, 2H), 1.40-1.33 (m, 2H), 0.87 (t, J =
7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 181.21 (s), 172.65 (s), 159.27 (s), 149.08 (s),
145.09 (s), 140.47 (s), 139.05 (s), 135.67 (s), 130.28 (s), 129.53 (s), 123.33 (s),120.63 (s),
120.34 (s), 119.38 (s), 116.91 (s), 114.83 (s), 110.74 (s), 100.66 (s), 100.15 (s), 99.85 (s),
97.30 (s), 66.49 (s), 57.01 (s), 55.17 (s), 53.54 (s), 48.57 (s), 44.78 (s). MS(ESI):
457.2016 (C29H29O5, [M+H]+).
(E)-2-(2-(4-butoxystyryl)-4,6-dimethoxyphenyl)-6-methoxy-4H-chromen-4-one (5r). Yellow
solid, Yield: 78%, Mp: 169-171 °C. IR (ATR Diamond Crystal, cm-1): v 2959, 2900, 2869,
1637, 1592, 1568, 1334, 1198, 1153, 1076, 1016, 849, 826; 1H NMR (600 MHz, DMSO) δ
7.57 (dd, J = 9.1, 1.2 Hz, 1H), 7.45 (d, J = 2.9 Hz, 1H), 7.36 (ddd, J = 9.1, 3.0, 1.3 Hz, 1H),
7.32 (d, J = 7.8 Hz, 2H), 7.23 (d, J = 16.1 Hz, 1H), 7.00 (s, 1H), 6.85-6.80 (m, 3H), 6.62 (s,
1H), 6.26 (s, 1H), 3.91-3.89 (m, 2H), 3.88 (d, J = 0.7 Hz, 3H), 3.86 (d, J = 1.0 Hz, 3H),
3.75 (s, 3H), 1.65-1.59 (m, 2H), 1.36 (tt, J = 13.7, 3.7 Hz, 2H), 0.87 (td, J = 7.4, 1.3
Hz,3H). 13C NMR (101 MHz, CDCl3): δ 186.21 (s), 171.65 (s), 162.87 (s), 160.24 (s),
157.29 (s), 143.98 (s), 141.75 (s), 139.82 (s), 135.28 (s), 134.13 (s), 129.36(s), 128.94 (s),
127.18 (s), 124.41 (s), 123.45 (s), 122.63 (s), 119.54 (s), 114.86 (s), 110.05 (s), 101.75 (s),
99.90 (s), 65.49 (s), 56.51 (s), 52.57 (s), 50.12 (s),48.02 (s), 44.57 (s),14.78 (s). MS(ESI):
487.2115 (C30H31O6 , [M+H]+ ).
(E)-6-bromo-2-(2-(4-butoxystyryl)-4,6-dimethoxyphenyl)-4H-chromen-4-one (5s). Yellow
solid, Yield: 79%, Mp: 265-267 °C. IR (ATR Diamond Crystal, cm-1): v 2959, 2900, 1652,
1
1597, 1567, 1459, 1331, 1248, 1160, 1082, 963, 797; H NMR (600 MHz, DMSO) δ 8.14
(d, J = 2.3 Hz, 1H), 7.93-7.90 (m, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H),
7.24 (d, J = 16.1 Hz, 1H), 6.99 (d, J = 16.5 Hz, 1H), 6.87 (t, J = 12.4 Hz, 1H), 6.83 (d, J =
8.5 Hz, 2H), 6.62 (s, 1H), 6.34 (s, 1H), 3.90 (dd, J = 10.9, 4.3 Hz, 2H), 3.88 (s, 3H), 3.75 (s,
3H), 1.66-1.60 (m, 2H), 1.41-1.34 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz,
17

ACCEPTED MANUSCRIPT
CDCl3): δ 181.91 (s), 162.65 (s), 157.27 (s), 156.08 (s), 155.09 (s), 140.48 (s), 139.05 (s),
138.67 (s), 137.28 (s), 130.53 (s), 128.63 (s), 128.94 (s), 127.08 (s), 121.91 (s), 120.86 (s),
120.13 (s), 118.74 (s), 115.16 (s), 110.65 (s), 105.35 (s), 98.90 (s), 66.19 (s), 57.11 (s),
55.27 (s), 53.62 (s), 52.12 (s), 15.28 (s). MS(ESI): 557.0834 (C29H27BrO5Na, [M+Na]+ ).
(E)-2-(2-(4-(benzyloxy)styryl)-4,6-dimethoxyphenyl)-4H-chromen-4-one (5t). Yellow solid,
Yield: 92%, Mp: 156-157 °C. IR (ATR Diamond Crystal, cm-1): v 2970, 2900, 1656, 1595,
1572, 1462, 1315, 1215, 1172, 1079, 830, 776; 1H NMR (600 MHz, DMSO) δ 8.08 (d, J =
7.9 Hz, 1H), 7.80-7.74 (m, 1H), 7.59 (t, J = 9.9 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.36 (dt,
J = 14.2, 7.5 Hz, 6H), 7.29 (t, J = 7.2 Hz, 1H), 7.24 (d, J = 16.1 Hz, 1H), 7.01 (s, 1H), 6.92
(d, J = 8.6 Hz, 2H), 6.88 (d, J = 16.1 Hz, 1H), 6.63 (s, 1H), 6.28 (s, 1H), 5.73 (s, 1H),
13
5.08-5.01 (m, 2H), 3.89 (s, 3H), 3.75 (s, 3H). C NMR (101 MHz, CDCl3 ): δ 181.91 (s),
162.65 (s), 160.27 (s), 159.48 (s), 157.09 (s), 156.48 (s), 152.05 (s), 151.67 (s), 145.28
(s),140.05 (s), 139.67 (s), 136.28 (s), 131.53 (s), 129.33 (s), 128.04 (s), 127.98 (s), 122.91
(s), 122.86 (s), 120.63 (s), 117.74 (s), 114.66 (s), 110.75 (s), 101.35 (s), 97.90 (s), 63.49 (s),
56.01 (s), 55.57 (s). MS(ESI): 491.1853 (C32H27O5 , [M+H]+).
(E)-2-(2-(4-(benzyloxy)styryl)-4,6-dimethoxyphenyl)-6-methoxy-4H-chromen-4-one (5u).
Yellow solid, Yield: 76%, Mp: 231-232 °C. IR (ATR Diamond Crystal, cm-1): v 2969, 2900,
1
1635, 1570, 1486, 1452, 1337, 1076, 1030, 968, 847, 697; H NMR (600 MHz, DMSO) δ
7.57 (d, J = 9.1 Hz, 1H), 7.45 (d, J = 3.1 Hz, 1H), 7.38 (dd, J = 10.1, 5.2 Hz, 3H),
7.36-7.31 (m, 4H), 7.29 (t, J = 7.0 Hz, 1H), 7.24 (d, J = 16.1 Hz, 1H), 7.00 (d, J = 2.0 Hz,
1H), 6.92 (d, J = 8.8 Hz, 2H), 6.86-6.81 (m, 1H), 6.62 (d, J = 2.1 Hz, 1H), 6.26 (s, 1H),
13
5.04 (d, J = 8.0 Hz, 2H), 3.88 (s, 3H), 3.86 (s, 3H), 3.75 (s, 3H). C NMR (101 MHz,
CDCl3): δ 181.67 (s), 163.72 (s), 160.62 (s), 158.98 (s), 157.19 (s), 156.36 (s), 152.17 (s),
150.81 (s), 145.28 (s), 140.05 (s), 138.82 (s), 136.28 (s), 131.53 (s), 129.33 (s), 128.04 (s),
127.98 (s), 122.91 (s), 122.86 (s), 120.63 (s), 117.74 (s), 114.66 (s), 110.75 (s), 101.35 (s),
97.90 (s), 66.52 (s), 56.91 (s), 53.61 (s), 45.91. MS(ESI): 521.1959 (C33H29O6, [M+H]+).
(E)-2-(2-(4-(benzyloxy)styryl)-4,6-dimethoxyphenyl)-6-bromo-4H-chromen-4-one
(5v).
18

ACCEPTED MANUSCRIPT
Yellow solid, Yield: 78%, Mp: 259-260 °C. IR (ATR Diamond Crystal, cm-1): v 2988, 2900,
1654, 1597, 1453, 1393, 1239, 1084, 974, 810; 1H NMR (600 MHz, DMSO) δ 8.14 (d, J =
2.5 Hz, 1H), 7.93 (dd, J = 8.9, 2.5 Hz, 1H), 7.59 (t, J = 11.9 Hz, 1H), 7.41-7.37 (m, 4H),
7.35 (t, J = 7.5 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 7.24 (d, J = 16.1 Hz, 1H), 7.00 (t, J = 6.8
Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 16.2 Hz, 1H), 6.62 (d, J = 2.0 Hz, 1H), 6.33
(s, 1H), 5.73 (s, 1H), 5.06 (s, 2H), 3.88 (s, 3H), 3.75 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ 182.69 (s), 164.92 (s), 161.82 (s), 157.54 (s), 157.01 (s), 156.36 (s), 152.17 (s), 150.81
(s), 145.28 (s),140.05 (s), 138.92 (s), 136.28 (s), 131.53 (s), 129.33 (s), 128.14 (s), 127.26
(s), 122.73 (s), 122.86 (s), 121.63 (s), 117.52 (s), 114.74 (s), 110.16 (s), 101.54 (s), 100.83
(s), 67.62 (s), 55.73 (s), 50.96 (s). MS(ESI): 591.0778 (C32H25BrO5Na, [M+Na]+).
4.4 Synthesis of resveratrol-based flavonol derivatives (7a~7o)
In a 50 mL round-bottom flask, a solution of compounds 6a~6o (1 mmol, 1.0 equiv) in
methanol was added NaOH (5 mmol, 5.0 equiv) and 30% H2O2 (5 mmol, 5.0 equiv), the
mixture was stirred at 40 °C until the reaction was completed. The reaction was cooled
back to room temperature and added ice-water, it was added hydrochloric acid to adjust pH
to neutral. The suspension was then stirred and vacuum filtered to provide the residue, the
crude product was purified by column chromatography on silica gel to obtained the desired
compounds.
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-4H-chromen-4-one
(7a).
White solid, Yield: 76%, Mp: 230-232 °C. 1H NMR (600 MHz, DMSO): δ 8.85 (s, 1H),
8.15 (d, J= 8.0 Hz, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.5
Hz, 1H), 7.30 (d, J = 8.6 Hz, 2H), 7.25 (d, J = 16.2 Hz, 1H), 7.03 (s, 1H), 6.84 (d, J = 8.6
Hz, 2H), 6.74 (d, J = 16.2 Hz, 1H), 6.61 (s, 1H), 3.89 (s, 3H), 3.72 (t, J = 20.0 Hz, 6H). 13C
NMR (151 MHz, DMSO): δ 175.66 (s), 164.97 (s), 162.31 (s), 161.96 (s), 158.47 (s),
148.85 (s), 143.43 (s), 141.68 (s), 136.50 (s), 133.97 (s), 132.35 (s), 131.01 (s), 128.08 (s),
127.54 (s), 125.75 (s), 125.28 (s), 121.55 (s), 117.30 (s), 114.78 (s), 104.04 (s), 100.93 (s),
59.01 (s), 58.66 (s), 58.19 (s). MS (ESI): 431.1483.(C26H22O6, [M+H]+).
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(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-7-methyl-4H-chromen-4-one
(7b). White solid, Yield: 78%, Mp: 210-213 °C. 1H NMR (600 MHz, DMSO): δ 8.77 (s,
1H), 8.02 (d, J = 8.2 Hz, 1H), 7.40 (s, 1H), 7.32 – 7.23 (m, 4H), 7.02 (s, 1H), 6.84 (d, J =
8.6 Hz, 2H), 6.71 (d, J = 16.2 Hz, 1H), 6.60 (s, 1H), 3.89 (s, 3H), 3.71 (t, J = 14.1 Hz, 6H),
2.42 (s, 3H). 13C NMR (101 MHz, DMSO): δ 172.37 (s), 161.80 (s), 159.18 (s), 158.83 (s),
155.49 (s), 145.24 (s), 144.25 (s), 140.12 (s), 138.50 (s), 130.77 (s), 129.23 (s), 127.85 (s),
125.93 (s), 124.72 (s), 122.64 (s), 119.93 (s), 117.87 (s), 100.88 (s), 97.79 (s), 114.18 (s),
111.76 (s), 55.85 (s), 55.51 (s), 55.05 (s), 21.15 (s). MS(ESI): 445.1645.(C27H24O6,
[M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-6-methyl-4H-chromen-4-one
(7c). White solid, Yield: 44%, Mp: 201-204 °C. 1H NMR (600 MHz, DMSO): δ 8.80 (s,
1H), 7.93 (s, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 8.5 Hz,
2H), 7.25 (d, J = 16.2 Hz, 1H), 7.02 (s, 1H), 6.84 (d, J = 8.3 Hz, 2H), 6.69 (d, J = 16.2 Hz,
13
1H), 6.60 (s, 1H), 3.89 (s, 3H), 3.70 (d, J = 13.1 Hz, 6H), 2.44 (s, 3H). C NMR (101
MHz, CDCl3): δ 172.94(s), 162.46 (s), 159.59 (s), 159.29 (s), 154.69 (s), 144.74 (s),
139.83 (s), 139.54 (s), 134.80 (s), 134.32 (s), 131.18 (s), 129.63 (s), 128.04 (s), 124.57 (s),
123.31 (s), 121.01 (s), 118.44 (s), 114.09 (s), 111.35 (s), 101.26 (s), 98.00 (s), 56.03 (s),
55.73 (s), 55.43 (s), 20.98 (s). MS(ESI): 445.1642.(C27H24O6, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-6-methoxy-4H-chromen-4-one
(7d). White solid, Yield: 89%, Mp: 188-191 °C. 1H NMR (600 MHz, DMSO): δ 8.81 (s,
1H), 7.56 (d, J = 9.1 Hz, 1H), 7.48 (d, J = 2.7 Hz, 1H), 7.34 (dd, J = 9.1, 2.7 Hz, 1H), 7.29
(d, J = 8.4 Hz, 2H), 7.25 (d, J = 16.2 Hz, 1H), 7.02 (s, 1H), 6.84 (d, J = 8.4 Hz, 2H), 6.69
(d, J = 16.2 Hz, 1H), 6.60 (s, 1H), 3.88 (d, J = 8.5 Hz, 6H), 3.70 (d, J = 14.7 Hz, 6H). 13C
NMR (101 MHz, DMSO): δ 172.09 (s), 161.82 (s), 159.19 (s), 158.83 (s), 155.84 (s),
150.32 (s), 145.61 (s), 139.86 (s), 138.53 (s), 130.79 (s), 129.22 (s), 127.83 (s), 123.18 (s),
122.70 (s), 122.63 (s), 120.05 (s), 114.18 (s), 111.75 (s), 103.96 (s), 100.92 (s), 97.80 (s),
55.86 (s), 55.69 (s), 55.51 (s), 55.05 (s). MS(ESI): 461.1591.(C27H24O7, [M+H]+).
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(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-7-fluoro-3-hydroxy-4H-chromen-4-one
(7e). White solid, Yield: 63%, Mp: 236-239 °C. 1H NMR (600 MHz, DMSO): δ 8.94 (s,
1H), 8.20 (dd, J = 8.9, 6.5 Hz, 1H), 7.58 (dd, J = 9.7, 2.1 Hz, 1H), 7.37-7.34 (m, 1H), 7.33
(d, J = 8.6 Hz, 2H), 7.26 (d, J = 16.2 Hz, 1H), 7.03 (d, J = 1.6 Hz, 1H), 6.84 (d, J = 8.7 Hz,
2H), 6.74 (d, J = 16.2 Hz, 1H), 6.60 (d, J = 1.7 Hz, 1H), 3.89 (s, 3H), 3.71 (d, J = 13.5 Hz,
6H). 13C NMR (101 MHz, CDCl3): δ 172.46 (s), 167.96 (s), 164.28 (s), 162.62 (s), 159.67
(s), 159.29 (s), 157.37 (s), 145.24 (s), 139.81 (s), 139.62 (s), 131.43 (s), 129.57 (s), 128.17
(s), 123.16 (s), 118.43 (s), 114.13 (s), 110.84 (s), 105.15 (s), 104.90 (s), 101.34 (s), 97.95
(s), 56.01 (s), 55.56 (s), 55.30 (s). MS(ESI): 449.1392.(C26H21FO6, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-6-fluoro-3-hydroxy-4H-chromen-4-one
(7f). White solid, Yield: 68%, Mp: 190-193 °C. 1H NMR (600 MHz, DMSO): δ 8.99 (s,
1H), 7.80 (dd, J = 22.0, 16.1 Hz, 1H), 7.72 (dd, J = 8.9, 3.7 Hz, 1H), 7.64 (t, J = 7.1 Hz,
1H), 7.32 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 16.2 Hz, 1H), 7.01 (d, J = 26.2 Hz, 1H), 6.82 (t,
J = 16.7 Hz, 2H), 6.72 (d, J = 16.2 Hz, 1H), 6.61 (s, 1H), 3.89 (s, 3H), 3.70 (d, J = 15.6
Hz,6H). 13C NMR (101 MHz, DMSO): δ 171.91 (s), 161.93 (s), 159.20 (s), 158.84 (s),
157.16 (s), 151.85 (s), 146.32 (s), 140.04 (s), 138.63 (s), 130.92 (s), 129.21 (s), 127.95 (s),
123.16 (s), 122.56 (s), 121.74 (s), 121.20 (s), 114.14 (s), 111.44 (s), 109.12 (s), 100.95 (s),
97.78 (s), 55.88 (s), 55.52 (s), 55.05 (s). MS(ESI): 449.1392.(C26H21FO6, [M+H]+).
(E)-8-chloro-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-4H-chromen-4-one
1
(7
g
). White solid, Yield: 55%, Mp: 208-210 °C. H NMR (600 MHz, DMSO): δ 9.07 (s,
1H), 8.11 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.34 (d, J =
8.5 Hz, 2H), 7.27 (d, J = 16.2 Hz, 1H), 7.04 (s, 1H), 6.84 (d, J = 8.5 Hz, 2H), 6.79 (d, J =
13
16.2 Hz, 1H), 6.63 (s, 1H), 3.90 (s, 3H), 3.74 (s, 3H), 3.70 (s, 3H). C NMR (151 MHz,
DMSO): δ 175.28 (s), 165.16 (s), 162.33 (s), 162.18 (s), 153.80 (s), 149.10 (s), 143.72 (s),
141.98 (s), 136.44 (s), 134.08 (s), 132.43 (s), 131.14 (s), 127.87 (s), 127.27 (s), 126.80 (s),
125.84 (s), 125.06 (s), 117.26 (s), 114.39 (s), 104.34 (s), 101.12 (s), 59.17 (s), 58.69 (s),
58.21 (s). MS(ESI): 465.1097.(C26H21ClO6, [M+H]+).
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(E)-7-chloro-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-4H-chromen-4-one
(7h). White solid, Yield: 48%, Mp: 209-210 °C. 1H NMR (600 MHz, DMSO): δ 8.98 (s,
1H), 8.14 (d, J = 8.7 Hz, 1H), 7.82 (d, J = 1.5 Hz, 1H), 7.50 (dd, J = 8.6, 1.7 Hz, 1H), 7.33
(d, J = 8.7 Hz, 2H), 7.25 (d, J = 16.2 Hz, 1H), 7.03 (d, J = 1.7 Hz, 1H), 6.84 (d, J = 8.7 Hz,
2H), 6.73 (t, J = 12.4 Hz, 1H), 6.60 (d, J = 1.8 Hz, 1H), 3.89 (s, 3H), 3.71 (d, J = 13.0 Hz,
6H). 13C NMR (101 MHz, CDCl3): δ 172.52 (s), 162.63 (s), 159.67 (s), 159.30 (s), 156.40
(s), 145.31 (s), 140.07 (s), 139.63 (s), 139.52 (s), 131.48 (s), 129.54 (s), 128.08 (s), 126.88
(s), 125.40 (s), 123.10 (s), 120.08 (s), 118.62 (s), 114.13 (s), 110.80 (s), 101.33 (s), 97.93
(s), 56.00 (s), 55.56 (s), 55.30 (s). MS(ESI): 465.1102.(C26H21ClO6, [M+H]+).
(E)-6-chloro-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-4H-chromen-4-one
(7i). White solid, Yield: 60%, Mp: 189-192°C. 1H NMR (600 MHz, CDCl3): δ 8.27 (s, 1H),
7.60 (d, J = 9.0 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 4.7 Hz, 3H), 7.06 (d, J =
16.0 Hz, 1H), 6.91 (s, 1H), 6.80 (d, J = 8.2 Hz, 2H), 6.77 (d, J = 16.1 Hz, 1H), 6.50 (s, 1H),
13
3.93 (s, 3H), 3.78 (d, J = 6.5 Hz, 6H). C NMR (101 MHz, CDCl3): δ 172.35 (s), 162.65
(s), 159.68 (s), 159.27 (s), 154.65 (s), 145.45 (s), 140.02 (s), 139.63 (s), 133.60 (s), 131.45
(s), 130.34 (s), 129.51 (s), 128.04 (s), 124.74 (s), 123.08 (s), 122.35 (s), 120.41 (s), 114.13
(s), 110.78 (s), 101.36 (s), 97.93 (s), 56.01 (s), 55.57 (s), 55.31(s). MS(ESI): 465.1101.
(C26H21ClO6, [M+H]+).
(E)-7-bromo-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-4H-chromen-4-one
(7j). White solid, Yield: 76%, Mp: 202-205°C. 1H NMR (600 MHz, DMSO): δ 9.03 (s,
1H), 8.05 (d, J = 8.6 Hz, 1H), 7.94 (d, J = 1.3 Hz, 1H), 7.63 (dd, J = 8.6, 1.1 Hz, 1H), 7.33
(d, J = 8.6 Hz, 2H), 7.25 (d, J = 16.2 Hz, 1H), 7.01 (d, J = 1.5 Hz, 1H), 6.83 (d, J = 8.6 Hz,
2H), 6.73 (d, J = 16.2 Hz, 1H), 6.59 (d, J = 1.6 Hz, 1H), 3.88 (s, 3H), 3.70 (d, J = 11.5 Hz,
6H). 13C NMR (101 MHz, CDCl3): δ 172.60 (s), 162.64 (s), 159.68 (s), 159.30 (s), 156.34
(s), 145.17 (s), 140.09 (s), 139.63 (s), 131.49 (s), 129.54 (s), 128.08 (s), 127.76 (s), 126.89
(s), 123.09 (s), 121.71 (s), 120.41 (s), 114.13 (s), 110.78 (s), 101.32 (s), 97.93 (s), 56.01 (s),
55.56 (s), 55.31 (s). MS(ESI): 509.0590.(C26H21BrO6, [M+H]+).
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(E)-6-bromo-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-4H-chromen-4-one
(7k). White solid, Yield: 82%, Mp: 201-204 °C. 1H NMR (600 MHz, DMSO): δ 9.08 (s,
1H), 8.22 (d, J = 2.4 Hz, 1H), 7.88 (dd, J = 8.9, 2.4 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.33
(d, J = 8.7 Hz, 2H), 7.26 (d, J = 16.2 Hz, 1H), 7.03 (d, J = 1.7 Hz, 1H), 6.84 (d, J = 8.7 Hz,
2H), 6.72 (d, J = 16.2 Hz, 1H), 6.60 (d, J = 1.8 Hz, 1H), 3.89 (s, 3H), 3.70 (d, J = 11.1 Hz,
6H). 13C NMR (101 MHz, CDCl3): δ 171.92 (s), 162.65 (s), 159.68 (s), 159.28 (s), 155.08
(s), 145.54 (s), 140.10 (s), 139.63 (s), 136.28 (s), 131.45 (s), 129.51 (s), 128.04 (s), 123.07
(s), 122.89 (s), 120.62 (s), 117.74 (s), 114.13 (s), 110.79 (s), 101.36 (s), 97.93 (s), 56.01 (s),
55.57 (s), 55.31 (s). MS(ESI): 511.0572.(C26H21BrO6, [M+H]+).
(E)-2-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-3-hydroxy-6,7-dimethyl-4H-chromen-4-
one (7l). White solid, Yield: 66%, Mp: 214-217 °C. 1H NMR (600 MHz, CDCl3): δ 8.03 (s,
1H), 7.27 (d, J = 6.3 Hz, 1H), 7.26 (s, 1H), 7.25 (s, 2H), 7.06 (d, J = 16.0 Hz, 1H), 6.91 (s,
1H), 6.82-6.77 (m, 3H), 6.50 (d, J = 1.4 Hz, 1H), 3.92 (d, J = 8.1 Hz, 3H), 3.77 (d, J = 3.9
13
Hz, 6H), 2.39 (d, J = 7.5 Hz, 6H). C NMR (101 MHz, CDCl3): δ 172.88 (s), 162.39 (s),
159.57 (s), 159.31 (s), 155.16 (s), 144.27 (s), 144.00 (s), 139.68 (s), 139.50 (s), 133.84 (s),
131.09 (s), 129.67 (s), 128.05 (s), 124.83 (s), 123.36 (s), 119.38 (s), 118.67 (s), 114.08 (s),
111.52 (s), 101.20 (s), 98.00 (s), 56.02 (s), 55.54 (s), 55.29 (s), 20.55 (s), 19.39 (s).
MS(ESI): 459.1803.(C28H26O6, [M+H]+).
(E)-2-(2-(4-ethoxystyryl)-4,6-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one (7m). White
solid, Yield: 77%, Mp: 244-247 °C. 1H NMR (600 MHz, CDCl3): δ 8.31 (d, J = 7.9 Hz,
1H), 7.66 (dd, J = 11.4, 4.2 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.26
(s, 1H), 7.24 (s, 1H), 7.07 (d, J = 16.1 Hz, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.81 (d, J = 16.2
Hz, 1H), 6.78 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 2.0 Hz, 1H), 6.29 (s, 1H), 3.99 (q, J = 7.0
Hz, 2H), 3.93 (s, 3H), 3.79 (s, 3H), 1.38 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3):
δ 173.15 (s), 162.51 (s), 159.31 (s), 159.01 (s), 156.42 (s), 145.02 (s), 139.93 (s), 139.63
(s), 133.32 (s), 131.35 (s), 129.45 (s), 128.06 (s), 125.54 (s), 124.38 (s), 123.14 (s), 121.55
(s), 118.69 (s), 114.64 (s), 111.22 (s), 101.30 (s), 97.96 (s), 63.47 (s), 56.02 (s), 55.55 (s),
14.79 (s). MS(ESI): 445.1642.(C27H24O6, [M+H]+).
23

ACCEPTED MANUSCRIPT
(E)-2-(2-(4-ethoxystyryl)-4,6-dimethoxyphenyl)-3-hydroxy-6-methoxy-4H-chromen-4-one
(7n). White solid, Yield: 81%, Mp: 220-223 °C. 1H NMR (600 MHz, CDCl3): δ 7.63 (d, J
= 3.0 Hz, 1H), 7.44-7.42 (m, 1H), 7.27 (dd, J = 9.2, 3.1 Hz, 1H), 7.24 (d, J = 8.7 Hz, 2H),
7.05 (d, J = 16.1 Hz, 1H), 6.91 (d, J = 2.1 Hz, 1H), 6.78 (dd, J = 12.4, 3.6 Hz, 3H), 6.50 (d,
J = 2.1 Hz, 1H), 6.28 (s, 1H), 3.99 (q, J = 7.0 Hz, 2H), 3.93 (d, J = 6.1 Hz, 6H), 3.78 (s,
3H), 1.38 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 172.71 (s), 162.47 (s),
159.29 (s), 159.00 (s), 156.41 (s), 151.58 (s), 144.80 (s), 139.62 (s), 139.54 (s),131.28 (s),
129.44 , 128.03 (s), 124.15 (s), 123.14 (s), 121.94 (s), 120.16 (s), 114.63 (s), 111.27 (s),
103.82 (s), 101.26 (s), 97.95 (s), 63.46 (s), 56.02 (s), 56.00 (s), 55.55 (s), 14.79 (s).
MS(ESI): 475.1747. (C28H26O7, [M+H]+).
(E)-6-bromo-2-(2-(4-ethoxystyryl)-4,6-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one
(7o). White solid, Yield: 78%, Mp: 200-205 °C. 1H NMR (600 MHz, CDCl3): δ 8.44 (d, J
= 2.4 Hz, 1H), 7.73 (dd, J = 9.0, 2.4 Hz, 1H), 7.39 (d, J = 8.9 Hz, 1H), 7.24 (d, J = 9.0 Hz,
2H), 7.06 (d, J = 16.0 Hz, 1H), 6.90 (d, J = 2.1 Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H), 6.76 (d, J
= 16.1 Hz, 1H), 6.50 (d, J = 2.1 Hz, 1H), 6.22 (s, 1H), 4.00 (q, J = 7.0 Hz, 2H), 3.93 (s,
3H), 3.78 (s, 3H), 1.38 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 171.91 (s),
162.65 (s), 159.27 (s), 159.08 (s), 155.09 (s), 145.48 (s), 140.05 (s), 139.67 (s), 136.28 (s),
131.53 (s), 129.33 (s), 128.04 (s), 127.98 (s), 122.91 (s), 122.86 (s), 120.63 (s), 117.74 (s),
114.66 (s), 110.75 (s), 101.35 (s), 97.90 (s), 63.49 (s), 56.01 (s), 55.57 (s), 14.78 (s).
MS(ESI): 525.0729.(C27H23BrO6, [M+H]+).
4.5 Cell culture
Mouse peritoneal macrophages were obtained from BeNa Culture Collection
Company. RAW264.7 cells were cultured in DMEM (Hyclone, USA) supplemented with
10% FBS (Biological Industries, Israel, 100 U/mL penicillin and100 µg/mL streptomycin
(Beyotime) at 37 °C in a humidified atmosphere containing 5% CO2.
4.6 Determination secretion of NO, TNF-α and IL-6
RAW264.7 cells were seeded into 48-well plate with 6×104 cells per well and
24

ACCEPTED MANUSCRIPT
incubated for 24ꢀh. RAW264.7 cells were pretreated with compounds (10 µM) for 1 h,
incubated with LPS (0.5 µg/mL) for 24 h. The supernatants were collected and examined
for NO production using Griess reagent (Beyotime, China). The levels of TNF-α and IL-6
in the culture medium were measured by ELISA (eBioScience, San Diego, CA).
4.7 Cell viability assay (MTT)
RAW264.7 cells were seeded into 96-well plate with 6×104 cells per well and
maintained at 37 °C in 5% CO2 about 24 h. RAW264.7 cells were pretreated with all
compounds (20 µM) for 1 h, incubated with LPS (0.5 µg/mL) for 24 h. After incubation at
37 °C for 4 h, the culture media containing MTT (prepared in PBS solution, 5 mg/mL)
were removed, and then DMSO (150 µL) was added into per well and the absorbance at
492 nm was measured by a microplate reader (MQX200, Bio-Tek, USA).
4.8 Western blotting
RAW264.7 cells were seeded into 96-well plate with 3×105 cells per well and
maintained at 37°C in 5% CO2 about 24 h. RAW264.7 cells were pretreated with
compound 7f (2, 1, 0.5 µM) for 1 h, incubated with LPS (0.5 µg/mL) for 0.5 h. The cells
were lysed in 300 µL RIPA cell lysis buffer (Contains PMSF and phosphatase inhibitors,
Beyotime china) and incubated on ice for 30 min. proteins were run on 12% SDS-PAGE
and then transferred to PVDF membrane (GE Healthcare, UK). The blotted membrane
incubated with specific primary antibodies (all antibody obtain from cell signaling
Technology, USA) overnight at 4°C. The membranes were washed in TBST (Beyotime
Biotech, Nantong, China), incubated with a 1:5000 dilutions of HRP-conjugated secondary
antibody (Beyotime Biotech, Nantong, China) for 1h at room temperature.
4.9 In vivo experiment
The 40 male C57BL/6 mice weighing 18~22 g were purchased from Animal
Department of Anhui Medical University. After one-week acclimatization, mice were
randomly divided into four groups on average, including the control group, LPS group,
compound 7f (10 mg/kg) +LPS group and compound 7f (20 mg/kg) +LPS group.
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30 min before a 20 mg/kg LPS tail vein injection. Control group animals were
received only an equal volume of saline by Intraperitoneal injection. Mice were
anesthetized and sacrificed 48 h after LPS injection. Lung tissues were harvested for
experiment. Lung tissue were fixed in 4% paraformaldehyde solution, embedded in
paraffin. After dehydration, sections were stained with hematoxylin and eosin (H&E) and
immunohistochemistry according to the previously reported method. The upper lung lobe
of the right lungs was excised, and obtain a wet weight. The lungs were then placed in an
oven at 60 °C for exceed 48 h until the dry weight was constant. Lung wet/dry weight ratio
was calculated to assess tissue edema. The lung tissue MPO activity was assessed using a
kit (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's
instruction. The research was approved by the Ethics Committee of Anhui Medical
University on the care.
4.10 Statistical analysis
Data are expressed as means±SEM and were analyzed statistically by analysis of
variance (ANOVE). A value of p<0.05 was considered to be statistically significant. All
experiments date were repeated at least three times.
Acknowledgments
The authors wish to thank the National Natural Science Foundation of China (No.
21572003, 21272008).
Supporting information
Spectral data of representative compounds containing 1H NMR, 13C NMR and HRMS.
CCDC-1551040 (compound 5r) and CCDC-1551039 (compound 5u) contain the
supplementary crystallographic data for this paper. These data can be obtained free of
charge via the URL http:// www.ccdc.cam.ac.uk/conts/retrieving. (or from the CCDC, 12
Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk). Tables S1~S11 and Figures S1~S15 are available free of charge.
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