Development of a novel nitric oxide (NO) production inhibitor with potential therapeutic effect on chronic inflammation

Article abstract of DOI:10.1016/j.ejmech.2020.112216

Inflammation is a complex biological response to stimuli. Activated macrophages induced excessively release of pro-inflammatory cytokines and mediators such as endogenous radical nitric oxide (NO) play a significant role in the progression of multiple inflammatory diseases. Both natural and synthetic chalcones possess a wide range of bioactivities. In this work, thirty-nine chalcones and three related compounds, including several novel ones, based on bioactive kava chalcones were designed, synthesized and their inhibitory effects on NO production in RAW 264.7 cells were evaluated. The novel compound (E)-1-(2′-hydroxy-4′,6′-dimethoxyphenyl)-3-(3-methoxy-4-(3-morpholinopropoxy)phenyl)prop-2-en-1-one (53) exhibited a better inhibitory activity (84.0%) on NO production at 10 μM (IC₅₀ = 6.4 μM) with the lowest cytotoxicity (IC₅₀ > 80 μM) among the tested compounds. Besides, western blot analysis indicated that compound 53 was a potent down-regulator of inducible nitric oxide synthase (iNOS) protein. Docking study revealed that compound 53 also can dock into the active site of iNOS. Furthermore, at the dose of 10 mg/kg/day, compound 53 could both significantly suppress the progression of inflammation on collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA) models. In addition, the structure-activity relationship (SAR) of the kava chalcones based analogs was also depicted.

Full text of DOI:10.1016/j.ejmech.2020.112216

Journal Pre-proof
Development of a novel nitric oxide (NO) production inhibitor with potential
therapeutic effect on chronic inflammation
Youzhe Yang, Zhe Wei, Alexander Tobias Teichmann, Frank Heinrich Wieland, Amu
Wang, Xiangui Lei, Yue Zhu, Jinxiang Yin, Tiantian Fan, Li Zhou, Chao Wang, Lijuan
Chen
PII:
S0223-5234(20)30183-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112216
EJMECH 112216
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 December 2019
Revised Date: 7 March 2020
Accepted Date: 7 March 2020
Please cite this article as: Y. Yang, Z. Wei, A.T. Teichmann, F.H. Wieland, A. Wang, X. Lei, Y. Zhu, J.
Yin, T. Fan, L. Zhou, C. Wang, L. Chen, Development of a novel nitric oxide (NO) production inhibitor
with potential therapeutic effect on chronic inflammation, European Journal of Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.ejmech.2020.112216.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Masson SAS.

Graphical abstract

Development of a novel nitric oxide (NO) production
inhibitor with potential therapeutic effect on chronic
inflammation
Youzhe Yang1,2,3,#, Zhe Wei2,#, Alexander Tobias Teichmann1, Frank Heinrich
Wieland1, Amu Wang4, Xiangui Lei4, Yue Zhu4, Jinxiang Yin4, Tiantian Fan3, Li
Zhou3, Chao Wang3, Lijuan Chen2,*
1
Sichuan Provincial Center for Gynaecology and Breast Diseases, The Affiliated Hospital of
Southwest Medical University, Luzhou 646000, P. R. China
2
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University and Collaborative Innovation Center, Chengdu 610041, P. R. China
3
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of
Sciences, Chengdu 610041, P. R. China
4
School of Science, Xihua University, Chengdu 610039, P. R. China
*
Correspondence: Prof. Lijuan Chen, Lab of Natural Product Drugs, State Key Laboratory of
Biotherapy and Cancer Center, West China Medical School, West China Hospital, Sichuan
University, Chengdu 610041, P. R. China. Tel.: +86-28-85164063. Fax: +86-28-85164060. E-mail:
chenlijuan125@163.com.
Abstract:
Inflammation is a complex biological response to stimuli. Activated
macrophages induced excessively release of pro-inflammatory cytokines and
mediators such as endogenous radical nitric oxide (NO) play a significant role in the
progression of multiple inflammatory diseases. Both natural and synthetic chalcones
possess a wide range of bioactivities. In this work, thirty-nine chalcones and three
related compounds, including several novel ones, based on bioactive kava chalcones
were designed, synthesized and their inhibitory effects on NO production in RAW
264.7
cells
were
evaluated.
The
novel
compound

(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3-methoxy-4-(3-morpholinopropoxy)p
henyl)prop-2-en-1-one (53) exhibited a better inhibitory activity (84.0 %) on NO
production at 10 µM (IC50 = 6.4 µM) with the lowest cytotoxicity (IC50 > 80 µM)
among the tested compounds. Besides, western blot analysis indicated that compound
53 was a potent down-regulator of inducible nitric oxide synthase (iNOS) protein.
Docking study revealed that compound 53 also can dock into the active site of iNOS.
Furthermore, at the dose of 10 mg/kg/day, compound 53 could both significantly
suppress the progression of inflammation on collagen-induced arthritis (CIA) and
adjuvant-induced arthritis (AIA) models. In addition, the structure-activity
relationship (SAR) of the kava chalcones based analogs was also depicted.
Keywords:
inflammation; nitric oxide (NO); kava chalcones; inducible nitric oxide synthase
(iNOS); structure-activity relationship (SAR)

1. Introduction
Inflammation is a primary response of organisms to various inflammatory agents,
such as bacteria, viruses, radiation and so on. The typical physiological features of
inflammation are characterized by redness, swelling, fever, pain, and even loss of
organ functions[1]. Previous studies indicated that activated macrophages induce
excessively release of pro-inflammatory cytokines and mediators such as endogenous
radical nitric oxide (NO), tumor necrosis factors-α (TNF-α), prostaglandin E2 (PGE2)
and interleukin-1β (IL-1β) play a significant role in the progression of multiple
inflammatory diseases[2-5]. The oxidation of L-arginine to L-citrulline producing NO
in the process is catalyzed by the inducible nitric oxide synthase (iNOS)[6, 7].
Pro-inflammatory cytokines and lipopolysaccharide (LPS) can activate the iNOS to
overproduce NO then further induce the injury at the inflammatory sites[8, 9]. Hence,
suppression of the expression and/or activity of iNOS and reduction the
overexpression of NO by drugs are crucial aspects of inflammation control[7, 10, 11].
Chalcones,
which
are
known
as
compounds
derived
from
1,3-diphenylprop-2-en-1-one with two aromatic rings linked by conjugated three
carbon α,β unsaturated carbonyl bridge (Figure 1), are a set of molecules that belong
to the flavonoid family[12, 13]. This class of molecules attracts considerable attention
and has been proved to possess a wide range of bioactivities such as anti-tumor,
antibacterial,
anti-tubercular,
anti-inflammatory,
anti-virus,
anti-malarial,
antileishmanial, antioxidant and so on[12, 14-20]. In order to enhance their
bioactivities and to develop potential therapeutic candidates, synthetic modifications
of chalcones have also been intensively carried out[21-25].
Kava, scientifically named as Piper methysticum, is an ancient perennial shrub
which is grown in the South Pacific. It is traditionally used for religious and
ceremonial events, medicinal purposes and social gatherings in this region[26-28].
The chemical constituents and their biological functions of kava extracts have been
extensively studied in recent decades. Chalcones and kavalactones are two major
phytochemicals present within this plant[29]. Kava chalcones (identified as FK A, B,

and C, Figure 1) from the kava extracts exhibit the activities of anti-tumor,
anti-inflammation and so on[30, 31]. Despite the increasing interest in the biological
profile of kava chalcones, little attention has been paid to the structural modifications
and structure-activity relationship of kava chalcones associated with their activities,
such as anti-inflammation[21, 31-34].
In the continuous effort to explore novel chemical entities for the treatment of
inflammation and related diseases[11, 35, 36], we were interested in designing and
synthesizing novel kava chalcones analogs. In this work, our design of target
compounds started from three points mainly based on scaffold-hopping method: ⅰ)
modification of B ring while keeping the special substitutions of A ring; ⅱ)
introduction of electron-donating and electron-withdrawing groups or atoms at the
middle double bond to interrupt the rigid plane structure of chalcone[37]; ⅲ)
modification of A ring to check the importance of the special substitutions. Then the
inhibitory effects on NO production of these compounds in LPS-induced RAW 264.7
cells were evaluated. Among these derivatives, a novel compound 53 was identified as
the most potent chemical molecule. The IC50 value of compound 53 on NO
production was 6.4 µM with much lower cytotoxicity than any other compound.
Western blot result revealed that compound 53 inhibited the expression of
LPS-induced iNOS protein in a dose-dependent manner. Besides, the inhibitory effect
was further confirmed by docking study which showed that compound 53 could bind
into the active site of iNOS. Furthermore, compound 53 significantly suppressed the
progression of inflammation in both two rheumatoid arthritis models of
collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA). The
structure-activity relationship (SAR) of these compounds was also discussed.
Figure 1. The structures of chalcone and FK A, FK B and FK C.
[Please Insert Figure 1 Here]

2. Results and discussion
2.1 Chemistry
Intermediates 5, 6, 7 were prepared from the commercially available reagent
m-trihydroxybenzene 1 in three steps. The target compounds were synthesized via
Claisen-Schmidt condensation between compounds 5, 6, 7, respectively, with
appropriate benzaldehydes (including purchased and self-prepared ones) under
suitable basic conditions with 30 % to 80 % isolated yields[22]. The general synthetic
route of FK A, FK B, FK C and 8 - 34 was illustrated in Scheme 1.
Scheme 1. The synthetic route of FK A, FK B, FK C, 8 - 34. Reagents and
o
conditions: a) Acetic anhydride or Propionic anhydride, BF3.OEt2, N2, 50 C; b)
Acetone, Me2SO4, K2CO3, reflux; c) dry CS2, Bromoacetyl bromide, AlCl3, N2, reflux;
d) dry MeCN, KF, TMBA(cat. amount), reflux; e) R = H or Me: MeOH, 40 % NaOH,
N2, 0 oC - r.t.; f) R = F: MeOH, Piperidine(cat. amount), r.t..
[Please Insert Scheme 1 Here]
In view of the preliminary activity data of series 1 - 3, compounds 35 - 39 were
designed and synthesized from compound 8 in various yields, shown as Scheme 2, to

check the importance of 2’-hydroxy group of A ring and α,β unsaturated carbonyl
bridge.
Scheme 2. The synthetic routes of 35 - 39. Reagents and conditions: a) reflux; b) dry
o
THF, NaH, N2, 0 C; c) MeOH, PtO2, r.t.; d) MeOH, c.c. HCl, reflux; e) MeOH,
pyridine, r.t..
By using the same method described above to synthesize FK A and so on, three
A ring simplified chalcones were obtained from 2’-hydroxy-4’-methoxyacetophenone
and
2’-hydroxyacetophenone
with
4-methoxybenzaldehyde
or
3-methoxybenzaldehyde. Their structures were outlined as the following Figure 2:
Figure 2. The structures of 40 - 42.
[Please Insert Figure 2 Here]
Taking the significance of a methoxy group at 3-position and potential helpful
contribution for the NO production inhibitive activity of a linker at 4-position on the
B ring into consideration, compounds 50 - 53 were designed and synthesized, the
preparative route was shown as Scheme 3.

Scheme 3. The synthetic route of 50 - 53. Reagents and conditions: a) MeCN, K2CO3,
reflux; b) MeCN, pyrrolidine or piperidine or N-methyl piperazine or morpholine,
K2CO3, r.t.; c) MeOH, 40 % NaOH, N2, 0 oC - r.t..
[Please Insert Scheme 3 Here]
2.2 Biological evaluation
2.2.1 Inhibition of NO production in LPS-induced RAW 264.7 cells
RAW 264.7 macrophages were treated with the target compounds at the
concentration of 10 µM and LPS (1 µg/mL) for 24 hours. Inhibitory effects of tested
compounds on NO production were indirectly measured by the analysis of nitrite
concentration based on the Griess reaction[35, 38]. A naturally occurring
anti-inflammatory flavone Luteolin (Lut.) was used as a positive control[39-42]. The
result was summarized in Table 1.
Table 1. Inhibitory effect of compounds FK A, FK B, FK C and 8 - 42, 50 - 53 on the production
of NO in LPS-induced RAW 264.7 macrophages.
Cpd.
FK A
FK B
FK C
8
NO inhibition rate (%) ± SDa
79.5 ± 2.6
Cpd.
28b
29b
30b
31b
32b
NO inhibition rate (%) ± SDa
50.3 ± 3.3
80.6 ± 2.4
58.1 ± 4.2
66.8 ± 1.3
47.4 ± 1.9
86.9 ± 2.2
70.0 ± 2.8
82.1 ± 3.7
50.8 ± 3.1
9

70.2 ± 3.1
35.4 ± 1.9
78.0 ± 2.4
70.6 ± 3.5
78.7 ± 3.6
70.0 ± 2.8
73.7 ± 4.5
67.6 ± 2.9
77.4 ± 4.5
78.4 ± 2.2
73.8 ± 1.0
30.8 ± 3.4
73.6 ± 2.4
78.2 ± 4.1
41.6 ± 3.9
68.3 ± 1.5
56.8 ± 2.7
57.5 ± 3.3
33b
34b
62.6 ± 3.8
42.6 ± 4.1
84.6 ± 3.2
85.6 ± 4.4
52.2 ± 1.9
57.3 ± 2.8
46.4 ± 3.7
70.0 ± 2.6
82.5 ± 1.8
76.7 ± 2.2
83.1 ± 2.2
74.8 ± 3.6
70.4 ± 3.1
84.0 ± 2.5
54.6 ± 2.9
100.0
10
11
12
13
14
15
16
17
18
19
20
21b
22b
23b
24
25b
26b
27
35b
36b
37b
38
39b
40
41
42
50b
51b
52b
53b
Lut.
Control
LPS
0.0
a The cells were pretreated with FK A, FK B, FK C and 8 - 42, 50 - 53, Lut. at a concentration of
10 µM and LPS (1 µg/mL) for 24 hours. The inhibition of the LPS-treated group was set as 0.0 %,
and the control group was set as 100.0 %. Inhibition rate (%) = 100.0 % – [Compound (OD540) –
Control (OD540)] / [LPS (OD540) – Control (OD540)] × 100.0 %. The results were shown as the
means ± SD (n = 3). b Novel compound.
2.2.2 IC50 of NO production and cytotoxicity of the selected compounds
To investigate the inhibitory effect on NO production in LPS stimulated RAW
264.7 macrophages of the derivatives in a dose-dependent manner, eight compounds
with the positive control Lut., including FK B, were chosen as excellent ones whose
NO inhibition rate at 10 µM was > 80.0 % in the previous study. The result expressed
in IC50 was shown as following:
Table 2. IC50 values of selected compounds on NO production in RAW 264.7 cells.

Cpd.
FK B
8
IC50 of NO production (µM)a
Cpd.
41
IC50 of NO production (µM)a
4.2
2.4
6.3
3.6
2.7
3.2
6.5
6.4
9.2
50
9
53
35
Lut.
36
a IC50 value is defined as the concentration that results in the reduction of 50.0 % production of
nitric oxide. All values are the mean of three independent experiments with the same patterns.
To check whether the inhibitory effect on the NO production was related to cell
viability or not, a MTT assay was adopted to determine the cytotoxicity of tested
compounds FK B, 8, 9, 35, 36, 41, 50, 53 and Lut.[35, 38]. The cytotoxicity of these
compounds was summarized in Table 3.
Table 3. Cytotoxicity of selected compounds in RAW 264.7 cells.
Cpd.
FK B
8
IC50 of cytotoxicity (µM)a
Cpd.
41
IC50 of cytotoxicity (µM)a
26.5
34.0
>40.0
30.7
34.0
29.7
>40.0
>80.0
>40.0
50
9
53
35
Lut.
36
a All values are the mean of three independent experiments with the same patterns.
2.2.3 Compound 53 inhibits iNOS expression in LPS-stimulated RAW 264.7 cell
Among the molecules tested, compound 53 was the most potent inhibitor of NO
production in LPS-induced macrophages with much lower cytotoxicity than any other
compound. Because the high level of NO is mainly generated by iNOS, and in order
to investigate the signaling pathway affected by compound 53[43], we studied the
inhibitory effects of compound 53 on iNOS expression in LPS-induced RAW 264.7 in
detail. Western blotting analysis indicated that the level of iNOS protein was
significantly decreased by compound 53 in a dose-dependent manner (Figure 3).
Therefore, compound 53 inhibited NO production through the down-regulation of

LPS-induced iNOS expression at the transcription level.
Figure 3. Effect of compound 53 on LPS-induced iNOS protein expression in RAW
264.7 cells. The RAW 264.7 cells were treated with LPS and different concentrations
of 53 for 24hr. Equal amounts of total protein were separated on 10 % SDS-PAGE
and blotted with antibodies (iNOS) as described in methods. Relative protein levels
were quantified by scanning densitometry. The values are expressed as means ± SD of
triplicate tests. (*p < 0.05, **p < 0.01)
[Please Insert Figure 3 Here]
2.2.4 Docking study of compound 53 with murine iNOS
In order to gain a better understanding of the binding mode between compound
53 with iNOS (PDB_ID: 3E67), a molecular docking study was carried out with the
Libdock program (Discovery Studio), and the result was shown in Figure 4.
Compound 53 could be divided into four parts, namely, A ring, B ring, propenone
linker and side chain with morpholine ring. According to the docking result, side
chain with morpholine ring occupied the polar domain and A ring dropped into the
non-polar part of iNOS. Both the oxygen atom as receptor and H atoms of methylenes
as a donor of morpholine ring formed a hydrogen bond with GLY365 and PRO344,
respectively. It was also noted that there was a Pi-alkyl interaction between the
morpholine ring with four pyrrole rings of the built-in ligand porphyrin ring of iNOS.
Moreover, a Pi-anion interaction between A ring with C-terminal of GLU488 and a
Pi-lone pair electron interaction between A ring with carbonyl group on ASN348 were
also observed. In summary, this computational result may help us to confirm our

hypothesis that compound 53 was a potent iNOS inhibitor through interaction with the
active site of iNOS.
Figure 4. Docking study of compound 53 with iNOS.
[Please Insert Figure 4 Here]
2.2.5. Anti-arthritic effects of compound 53 on CIA models
Collagen-induced arthritis (CIA) is a form of chronic arthritis and commonly
used to test agents for anti-inflammatory activity[44]. As the most potent molecule,
compound 53 was chosen to examine the ability of anti-inflammation on CIA models.
CIA mice were treated orally with compound 53 at a dose of 10 mg/kg/day following
the appearance of first CIA signs.
Results showed that the severity of CIA was alleviated in compound 53 treated
animal models (Figure 5). The performance of the arthritis score was significantly
reduced during the treatment (Figure 5A). Macroscopic observation of the ankle joint
and joint histology showed that compound 53 significantly reduced the paw redness
compared with the vehicle group, further confirming the therapeutic effect.
Histological evaluation by H&E staining and Safranin O staining revealed that a
broken joint structure was observed in the vehicle group with a large amount of
inflammatory cell infiltration and cartilage destruction (Figure 5B). However, mice
treated with compound 53 significantly inhibited these changes with few signs of
inflammation, and the histological state of the joint was intact and the cartilage was
almost completely preserved.
To explore whether compound 53 possesses the ability to reduce the expression
of pro-inflammatory cytokines, joint tissues were collected and the relative mRNA
levels of TNF-α, IL-lβ and IL-6 were measured by RT-PCR. It was found that the

levels of these pro-inflammatory cytokines in joint tissues were significantly reduced
in the compound 53 treated groups (Figure 5C).
According to reports, CD4 and CD8 double-positive T cells (DPT) in patients
with rheumatoid arthritis (RA) play an important role in the pathogenesis and
progression of RA[45, 46]. To analyze the expression level of DPT in CIA mice, the
flow cytometry method was used. The results showed that the expression of DPT in
compound 53 treated group mice was significantly decreased (Figure 5D). These
observations suggested that compound 53 might be an effective agent for the
management of RA.
Figure 5. Anti-arthritic effects of compound 53 on CIA models. Compound 53 was
orally administered once daily at a dose of 10 mg/kg. (A) Clinical score of the
compound 53 group. The results were expressed as the means (n = 5). (B)
Representative histopathologies of the knee joints stained with H&E and Safranin O
fast green (× 40). (C) Inhibitory effects on the mRNA levels of TNF-α, IL-1β and IL-6
in joint tissues. The mRNA levels were measured by RT-PCR. (D) Lymphocytes from
spleen and lymph nodes were collected after dissection. Cells were stained for CD4
and CD8. The percentage/mean fluorescence intensity of CD4+ and CD8+ cells were
presented in the upper right quadrant. One representative experiment of three
independent experiments was shown.
[Please Insert Figure 5 Here]

2.2.6 Anti-arthritic effects of compound 53 on AIA models
In most cases, adjuvant-induced arthritis (AIA) used for RA was also applied to
agents testing for anti-inflammatory activity. Compound 53 was chosen to verify the
anti-arthritis effects on AIA model for its effective inhibition on CIA model. Almost
all of the rats developed arthritis within 13 - 15 days after the adjuvant injection. It
was then treated with compound 53 at a dose of 10 mg/kg/day.
As shown in Figure 6, compound 53 treated rats did not show significant
arthritic symptoms, and arthritis scores were significantly reduced during the
treatment (Figure 6A). According to the results of H&E staining (Figure 6B), signs
of severe arthritis were shown in the vehicle group, while no obvious
histopathological changes were observed in the group treated with compound 53 at a
dose of 10 mg/kg/day. This result indicated compound 53 also possesses potential
immunomodulatory activity.
Figure 6. Anti-arthritic effects of compound 53 on AIA models. Compound 53 was
orally administered once daily at a dose of 10 mg/kg. (A) Clinical disease activity
score of compound 53. The result was expressed as the means (n = 5, ***p < 0.001).
(B) Representative histopathologies of the knee joints stained with H&E (× 40).
[Please Insert Figure 6 Here]
3. Structure-activity relationship (SAR) analysis
According to the results in Table 1, among kava chalcones, FK A and FK B
showed a better inhibitory effect on NO production than FK C (FK B 80.6 % ≈ FK A
79.5 % > FK C 66.8 %), indicated that the inhibitory effect was related to the
hydrogen bond formation ability or acidity of the B ring at 4-position. The
introduction of a single methoxyl group on the B ring at 3-position slightly increased

the inhibitory effect (8 86.9 % > FK B 80.6 % ≈ FK A 79.5 %). The modification of
the hydroxyl group on the A ring at 2’-position or removal of the methoxyl group at
6’-position of 8 did not decrease the effect distinctly (35 84.6 %, 36 85.6 %, 41
82.5 %). However, the removal of the methoxyl group on the A ring at 6’-position of
FK A, decreased the inhibitory effect slightly (FK A 79.5 % > 40 70.0 %). These
results indicated that A ring was slightly tolerant to simple modifications at specific
positions, and the methoxyl group at 3-position of the B ring was very critical to
maintain or increase the activity. The addition of one or two methoxyl groups or other
electron donating groups at different positions on the B ring did not show improved
inhibitory effects (FK A 79.5 % VS 14 78.7 %, 16 73.7 %, 17 67.6 %, 18 77.4 %, 19
78.4 %). The addition of electron withdrawing groups on the B ring decreased the
inhibitory effect obviously (FK B 80.6 % VS 10 70.2 %, 11 35.4 %; 14 78.7 % VS 20
73.8 %, 21 38.0 %). These results suggested that electron density on the B ring played
a crucial role. The replacement of benzene ring by heterocycle failed to increase the
inhibitory effect (FK B 80.6 % VS 12 78.0 %). Depressingly, the addition of a methyl
group or a fluorine atom at the α-position of the double bond decreased the inhibitory
effect dramatically (FK B 80.6 % VS 24 41.6 %, 28 50.3 %; 8 86.9 % VS 25 68.3 %,
29 58.1 %). Saturation of α, β-double bond (8 86.9 % VS 37 52.2 %), formation of
flavanone (8 86.9 % VS 38 57.3 %) or oxime (8 86.9 % VS 39 46.4 %) also have
obvious negative effects on the inhibitory activity. These results proved that the main
structure of chalcone with an unsaturated three carbon bridge was significant for
maintaining the anti-inflammatory activity. N.K. Sahu and co-workers also pointed
out the common unsaturated ketone moiety is likely to be responsible for the versatile
biological activities observed, as removal of this structural feature results in loss of
bioactivity[47]. At the early stage of our study, undesirable cytotoxicity of several
compounds including FK B, 8, 35, 36, 41 were detected according to the MTT assay
results. So, a further modification of these compounds should be carried out to reduce
the toxicity. The introduction of a hydroxyethyl or hydroxypropyl group to 4-position
of FK C increased the activity (23 78.2 % > 22 73.6 % > FK C 66.8 %) slightly
indicated that further modification on this position could be acceptable. Taking the

importance of a methoxyl group at the 3-position of B ring into consideration,
compounds 50, 51, 52, 53 were designed and synthesized. Fortunately, compound 53
showed a better activity (84.0 %) with much lower cytotoxicity than any other
compound. The structure-activity relationship of the kava chalcones analogs was
depicted in Figure 7.
Figure 7. Structure-activity relationship analysis.
[Please Insert Figure 7 Here]
H. ur Rashid and co-workers recently summarized the naturally occurring and
synthetic chalcones with various substitutions on the A ring and B ring as NO
production inhibitors in detail[14]. As is shown in Table 4, these chalcones bring new
structural elements that will help us in the design of more novel potential chalcones
and clarify the SAR of kava chalcones in future.
Table 4. Selected chalcones from different sources as NO inhibitor and their IC50 values.
Name of chalcone
source
IC
50
val
ue
(µ
M)
1-[6-(3,7-dimethyl-octa-2,6-dienyl)-5,7-dihydroxy-2,2-dimethyl-2H-chromen-8- Mallotu 7.6
yl]-3-(4-hydroxy-phenyl)-propenone
s
philippi
nensis

3-(3,4-dihydroxy-phenyl)-1-[6-(3,7-dimethyl-octa-2,6-dienyl)-5,7-dihydroxy-2,2 Mallotu 9.5
-dimethyl-2H-chromen-8-yl]-propenone
s
philippi
nensis
1-[5,7-dihydroxy-2-methyl-6-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-2H Mallotu 38.
-chromen-8-yl]-3-(3,4-dihydroxy-phenyl)-propenone
s
6
philippi
nensis
Broussochalcone A
Brousso 11.
netia
papyrife
ra
3
(L.)
Isobavachalcone
Bavachromene
Cullen
corylifol
ium
1.6
±
0.1
1
Cullen
corylifol
ium
2.4
±
0.1
8
Kanzonol B
Cullen
corylifol
ium
2.2
±
0.2
1
4-hydroxylonchocarpin
Psorale 10.
a
2
corylifol
ia

(E)-1-(2,6-dimethoxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one
(E)-1-(2,5-dimethoxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one
3,4,5-trimethoxy-4’-fluorochalcone
Syntheti 0.6
c
Syntheti 0.7
c
Syntheti 0.0
c
33
(3-(2-Hydroxyphenyl)-1-(thiophene-3-yl)prop-2-en-1-one) (TI-I-174)
2-(3-(3,4-dimethoxyphenyl)propyl)-5-methoxyphenol
Syntheti 5.7
c
5
Syntheti 6.5
c
(E)-1-(4-hydroxy-3-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one Syntheti 4.1
c
9
(E)-1-(3-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
2’-methoxy-3,4-dichlorochalcone
Syntheti 2.8
c
8
Syntheti 7.1
c
2’-hydroxy-6’-methoxychalcone
Syntheti 9.6
c
2’-hydroxy-3-bromo-6’-methoxychalcone
2’,5’-dihydroxy-4-chloro-dihydrochalcone
Syntheti 7.8
c
Syntheti 4.0
c
±
1.6
4. Materials and Methods
4.1. Chemistry
All the reagents and solvents of analytical grade were purchased from
commercial sources and used without further purification. Reactions were monitored
by thin-layer chromatography (TLC) on silica gel 60 F-254 thin-layer plates and spots
of desired products were located by ultraviolet (UV) lamp and Iodine. Intermediates

and target compounds were recrystallized from MeOH or purified by column
chromatography using 300 - 400 mesh silica gel. 1H spectra were recorded on Bruker
AV-600, 400 and 300 MHz, 13C spectra were recorded on Bruker AV-100 MHz.
HRMS (high resolution) spectra were measured by Waters Vion® IMS QTof mass
spectrometer utilizing electrospray ionization (ESI).
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
1
(FK A): yellowish powder, yield 65.0 %. H NMR (300 MHz, CDCl3): δ (ppm) =
3.83 (s, 3H), 3.85 (s, 3H), 3.91 (s, 3H), 5.96 (s, 1H), 6.10-6.11 (d, J = 3.0 Hz, 1H),
6.91-6.94 (d, J = 9.0 Hz, 2H), 7.55-7.58 (d, J = 9.0 Hz, 2H), 7.78 (s, 1H), 7.79 (s, 1H),
14.00 (s, 1H). The analytical data were nicely consistent with related reference[48].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-phenylprop-2-en-1-one (FK B):
1
yellowish powder, yield 71 %. H NMR (600 MHz, CDCl3): δ (ppm) = 3.84 (s, 3H),
3.92 (s, 3H), 5.97 (d, J = 2.3 Hz, 1H), 6.12 (d, J = 2.3 Hz, 1H), 7.40-7.41 (m, 3H),
7.60-7.61 (m, 2H), 7.78-7.80 (d, J = 18.0 Hz, 1H), 7.89-7.91 (d, J = 12.0 Hz, 1H).
The analytical data were nicely consistent with related reference[33].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(FK C): yellowish powder, yield 51 %. 1H NMR (600 MHz, CDCl3): δ (ppm) = 3.84
(s, 3H), 3.92 (s, 3H), 5.96-5.97 (d, J = 6.0 Hz, 1H), 6.11 (d, J = 6.0 Hz, 1H),
6.86-6.87 (d, J = 6.0 Hz, 1H), 7.07-7.08 (d, J = 6.0 Hz, 1H), 7.51-7.55 (q, 2H),
7.77-7.80 (q, 2H), 14.39 (s, 1H). The analytical data were nicely consistent with
related reference[48].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one
1
(8): yellowish powder, yield 74 %. H NMR (600 MHz, CDCl3): δ (ppm) = 3.84 (s,
3H), 3.85 (s, 3H), 3.92 (s, 3H), 5.96-5.97 (d, J = 6.0 Hz, 1H), 6.11-6.12 (d, J = 6.0 Hz,
1H), 6.93-6.95 (q, 1H), 7.13 (s, 1H), 7.20-7.22 (d, J = 12.0 Hz, 1H), 7.31-7.34 (t, J =
6.0, 12.0 Hz, 1H), 7.73-7.76 (d, J = 18.0 Hz, 1H), 7.86-7.89 (d, J = 18.0 Hz, 1H). The
analytical data were nicely consistent with related reference[49].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-p-tolylprop-2-en-1-one (9): yellowish
powder, yield 71 %. 1H NMR (600 MHz, CDCl3): δ (ppm) = 2.39 (s, 3H), 3.84 (s, 3H),
3.92 (s, 3H), 5.96-5.97 (d, J = 6.0 Hz, 1H), 6.11 (d, J = 6.0 Hz, 1H), 7.20-7.22 (d, J =

12.0 Hz, 2H), 7.50-7.51 (d, J = 6.0 Hz, 2H), 7.76-7.78 (d, J = 12.0 Hz, 1H), 7.86-7.88
(d, J = 12.0 Hz, 1H). The analytical data were nicely consistent with related
reference[50].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(4-bromophenyl)prop-2-en-1-one
(10): yellowish powder, yield 45 %. 1H NMR (300 MHz, CDCl3): δ (ppm) = 3.84 (s,
3H), 3.92 (s, 3H), 5.95-5.97 (d, J = 6.0 Hz, 1H), 6.10-6.12 (d, J = 6.0 Hz, 1H),
7.44-7.47 (d, J = 9.0 Hz, 2H), 7.52-7.55 (d, J = 9.0 Hz, 2H), 7.67-7.72 (d, J = 15.0 Hz,
1H), 7.65-7.90 (d, J = 15.0 Hz, 1H), 14.21 (s, 1H). The analytical data were nicely
consistent with related reference[21].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(4-nitrophenyl)prop-2-en-1-one (11):
1
brown powder, yield 57 %. H NMR (600 MHz, DMSO-d6): δ (ppm) = 3.90 (s, 3H),
3.93 (s, 3H), 6.37-6.38 (d, J = 6.0 Hz, 1H), 6.70-6.70 (d, 1H), 6.83 (s, 1H), 8.13-8.15
(d, J = 12.0 Hz, 2H), 8.28-8.30 (d, J = 12.0 Hz, 2H). The analytical data were nicely
consistent with related reference[51].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-one (12):
brown powder, yield 44 %. 1H NMR (600 MHz, CDCl3): δ (ppm) = 3.87 (s, 3H), 3.94
(s, 3H), 5.99-6.00 (d, J = 6.0 Hz, 1H), 6.14 (d, 1H), 7.37-7.38 (t, 1H), 7.74-7.77 (d, J
= 18.0 Hz, 1H), 7.89-7.90 (d, J = 6.0 Hz, 1H), 7.96-7.99 (d, J = 18.0 Hz, 1H), 8.63 (s,
1H), 8.87 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 191.84, 182.51, 165.81, 165.18,
161.88, 136.95, 134.90, 130.91, 125.37, 110.56, 106.36, 93.85, 91.14, 62.16, 56.28,
55.74. ESI-HRMS calculated for C16H16NO4 [M+H]+: 286.1001, found: 286.1070.
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)prop-
1
2-en-1-one (13): light yellowish powder, yield 66 %. H NMR (600 MHz, CDCl3): δ
(ppm) = 3.84 (s, 3H), 3.92 (s, 3H), 3.94 (s, 3H), 5.96-5.97 (d, J = 6.0 Hz, 1H), 6.11 (d,
1H), 6.86-6.88 (d, J = 12.0 Hz, 1H), 7.09-7.11 (q, J = 6.0, 6.0 Hz, 1H), 7.24-7.26 (d, J
= 12.0 Hz, 1H), 7.71-7.74 (d, J = 18.0 Hz, 1H), 7.76-7.79 (d, J = 18.0 Hz, 1H). The
analytical data were nicely consistent with related reference[52].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-o
ne (14): yellowish powder, yield 82 %. 1H NMR (600 MHz, CDCl3): δ (ppm) = 3.84
(s, 3H), 3.91 (s, 3H), 3.93 (s, 3H), 3.94 (s, 3H), 5.97-5.97 (d, J = 6.0 Hz, 1H),

6.10-6.12 (d, J = 6.0 Hz, 1H), 6.91 (s, 1H), 7.13 (s, 1H), 7.24 (s, 1H), 7.77-7.79 (q,
2H). The analytical data were nicely consistent with related references[53, 54].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(benzo[d][1,3]dioxol-5-yl)prop-2-en-
1
1-one (15): yellowish powder, yield 49 %. H NMR (600 MHz, CDCl3): δ (ppm) =
3.84 (s, 3H), 3.92 (s, 3H), 5.96-5.97 (d, J = 6.0 Hz, 1H), 6.02 (s, 2H), 6.11-6.11 (d, J =
6.0 Hz, 1H), 6.83-6.84 (d, J = 6.0 Hz, 1H), 7.09-7.10 (d, J = 6.0 Hz, 1H), 7.12 (s, 1H),
7.74-7.74 (d, 2H). The analytical data were nicely consistent with related
reference[49].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(2,5-dimethoxyphenyl)prop-2-en-1-o
ne (16): yellowish powder, yield 59 %. 1H NMR (600 MHz, CDCl3): δ (ppm) = 3.82
(s, 3H), 3.84 (s, 3H), 3.87 (s, 3H), 3.90 (s, 3H), 5.96 (d, J = 2.2 Hz, 1H), 6.11 (d, J =
2.2 Hz, 1H), 6.87 (d, J = 9.0 Hz, 1H), 6.92 (dd, J = 9.0, 3.0 Hz, 1H), 7.15 (d, J = 2.9
Hz, 1H), 7.93 (d, J = 18.0 Hz, 1H), 8.09 (d, J = 18.0 Hz, 1H). The analytical data
were nicely consistent with related reference[55].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-o
1
ne (17): yellowish powder, yield 68 %. H NMR (600 MHz, CDCl3): δ (ppm) =
3.83-3.91 (s, 12H), 5.96 (d, J = 3.0 Hz, 1H), 6.11 (d, J = 3.0 Hz, 1H), 6.47 (d, J = 3.0
Hz, 1H), 6.53-6.54 (dd, J = 3.0, 3.0 Hz, 1H), 7.54 (d, J = 12.0 Hz), 7.90 (d, J = 18.0
Hz, 1H), 8.11 (d, J = 18.0 Hz, 1H). The analytical data were nicely consistent with
related reference[55].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-
1
1-one (18): brown powder, yield 78 %. H NMR (600 MHz, CDCl3): δ (ppm) =
3.84-3.91 (s, 15H), 5.97 (s, 1H), 6.12 (s, 1H), 6.84 (s, 2H), 7.70-7.72 (t, J = 6.0, 6.0
Hz, 1H), 7.77-7.81 (q, J = 6.0, 6.0 Hz, 1H). The analytical data were nicely consistent
with related references[56, 57].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-
1-one (19): dark yellowish powder, yield 81 %. 1H NMR (300 MHz, CDCl3): δ (ppm)
= 3.82-3.94 (s, 15H), 5.95 (d, J = 3.0 Hz, 1H), 6.10 (d, J = 3.0 Hz, 1H), 6.51 (s, 1H),
7.11 (s, 1H), 7.85 (d, J = 15.5 Hz, 1H), 8.13 (d, J = 15.5 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ 192.81, 168.38, 165.82, 162.38, 154.52, 152.24, 143.19, 138.01,

125.28, 116.19, 111.44, 106.44, 96.87, 93.82, 91.20, 56.48, 56.37, 56.08, 55.73, 55.58.
The analytical data were nicely consistent with related references [58].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3-bromo-4-methoxyphenyl)prop-2-
1
en-1-one (20): dark yellowish powder, yield 29 %. H NMR (300 MHz, CDCl3): δ
(ppm) = 3.84 (s, 3H), 3.92 (s, 3H), 3.94 (s, 3H), 5.96-5.97 (d, J = 3.0 Hz, 1H),
6.10-6.11 (d, J = 3.0 Hz, 1H), 6.90-6.93 (d, J = 9.0 Hz, 1H), 7.48-7.51 (m, 1H),
7.52-7.82 (m, 3H), 14.30 (s, 1H). The analytical data were nicely consistent with
related reference[59].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3,4-dimethoxy-5-nitrophenyl)prop-
2-en-1-one (21): brown powder, yield 68 %. 1H NMR (600 MHz, CDCl3): δ (ppm) =
3.91 (s, 3H), 3.96 (s, 3H), 3.99 (s, 3H), 4.05 (s, 3H), 6.15 (s, 1H), 6.28 (s, 1H), 7.32 (s,
2H), 7.60 (s, 1H), 7.64 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 179.80, 169.22, 168.87,
159.65, 152.46, 149.01, 148.68, 142.12, 121.87, 113.25, 108.12, 105.22, 105.05,
94.22, 89.31, 56.51, 56.46, 56.29, 56.25.
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(4-(2-hydroxyethoxy)phenyl)prop-2-
1
en-1-one (22): pale yellowish powder, yield 78 %. H NMR (300 MHz, CDCl3): δ
(ppm) = 3.81 (s, 3H), 3.83 (s, 3H), 3.97-4.01 (m, 2H), 4.12-4.15 (t, J = 3.0, 6.0 Hz,
2H), 5.96-5.97 (d, J = 3.0 Hz, 1H), 6.10-6.11 (d, J = 3.0 Hz, 1H), 6.93-6.96 (d, J = 9.0
Hz, 2H), 7.55-7.58 (d, J = 9.0 Hz, 2H), 7.79-7.80 (d, J = 3.0 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 192.57, 168.38, 166.06, 162.45, 142.28, 130.14, 128.73, 125.38,
114.91, 93.80, 91.26, 69.29, 61.39, 55.87, 55.60. ESI-HRMS calculated for C19H21O6
[M+H]+: 345.1260, found: 345.1330.
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(4-(3-hydroxypropoxy)phenyl)prop-
1
2-en-1-one (23): pale yellowish powder, yield 77 %. H NMR (600 MHz, CDCl3): δ
(ppm) = 2.06-2.08 (t, J = 6.0, 6.0 Hz, 2H), 3.84 (s, 3H), 3.87-3.89 (t, J = 6.0, 6.0 Hz,
2H), 3.92 (s, 3H), 4.17-4.19 (t, J = 6.0, 6.0 Hz, 2H), 5.97-5.97 (d, J = 6.0 Hz, 1H),
6.11-6.11 (d, J = 6.0 Hz, 1H), 6.93-6.94 (d, J = 6.0 Hz, 2H), 7.55-7.56 (d, J = 6.0 Hz,
2H), 7.79 (d, 2H). 13C NMR (100 MHz, CDCl3) δ 192.60, 168.38, 168.01, 166.04,
162.46, 160.58, 142.45, 130.15, 128.43, 125.18, 119.53, 114.86, 110.49, 106.35,
103.88, 93.80, 91.25, 87.50, 65.66, 60.21, 55.87, 55.61, 31.92. ESI-HRMS calculated

for C20H22O6Na [M+Na]+: 381.1314, found: 381.1306.
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-2-methyl-3-(4-methoxyphenyl)prop-2-
1
en-1-one (24): light yellowish powder, yield 78 %. H NMR (600 MHz, CDCl3): δ
(ppm) = 2.15 (s, 3H), 3.71 (s, 3H), 3.83 (s, 6H), 5.95 (s, 1H), 6.14 (s, 1H), 6.76 (s,
1H), 6.91-6.92 (d, J = 12.0 Hz, 2H), 7.34-7.36 (d, J = 12.0 Hz, 2H), 13.56 (s, 1H).
The analytical data were nicely consistent with related reference[60].
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-2-methyl-3-(3-methoxyphenyl)prop-2-
1
en-1-one (25): light yellowish powder, yield 76 %. H NMR (300 MHz, CDCl3): δ
(ppm) = 2.14 (s, 3H), 3.74 (s, 3H), 3.82 (s, 3H), 3.84 (s, 3H), 5.94-5.95 (d, J = 3.0 Hz,
1H), 6.13-6.14 (d, J = 3.0 Hz, 1H), 6.68 (s, 1H), 6.82 (d, J = 3.0 Hz, 1H), 6.85 (d, J =
13
3.0 Hz, 1H), 6.91 (s, 1H), 7.30 (d, J = 6.0 Hz, 1H). C NMR (100 MHz, CDCl3) δ
201.83, 165.85, 165.56, 161.70, 159.20, 138.15, 132.73, 130.99, 129.21, 113.83,
105.51, 93.64, 91.28, 55.70, 55.60, 55.31, 15.41. ESI-HRMS calculated for C19H21O5
[M+H]+: 329.1311, found: 329.1386.
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-2-methyl-3-p-tolylprop-2-en-1-one
1
(26): light yellowish powder, yield 69 %. H NMR (600 MHz, CDCl3): δ (ppm) =
2.15 (s, 3H), 2.32 (s, 3H), 3.71 (s, 3H), 3.83 (s, 3H), 5.94-5.95 (d, J = 6.0 Hz, 1H),
6.13-6.14 (d, J = 6.0 Hz, 1H), 6.73 (s, 1H), 7.18-7.20 (d, J = 12.0 Hz, 2H), 7.28-7.29
13
(d, J = 6.0 Hz, 2H), 12.10 (s, 1H). C NMR (100 MHz, CDCl3) δ 202.01, 166.02,
165.81, 161.84, 139.37, 137.67, 133.76, 132.25, 129.40, 129.10, 105.42, 93.62, 91.29,
55.70, 55.61, 21.32, 15.40. ESI-HRMS calculated for C19H20O4 [M+H]+: 313.1362,
found: 313.1432.
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-2-methyl-3-phenylprop-2-en-1-one
1
(27): light yellowish powder, yield 66 %. H NMR (600 MHz, CDCl3): δ (ppm) =
2.15 (s, 3H), 3.73 (s, 3H), 3.84 (s, 3H), 5.95 (d, J = 3.0 Hz, 1H), 6.13 (d, J = 3.0 Hz,
1H), 6.73 (s, 1H), 7.27-7.39 (m, 5H), 12.23 (s, 1H). The analytical data were nicely
consistent with related reference[61].
(Z)-2-fluoro-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-e
n-1-one (28): yellowish powder, yield 67 %. 1H NMR (600 MHz, CDCl3): δ (ppm) =
3.86 (s, 3H), 3.91 (s, 3H), 3.96 (s, 3H), 6.13-6.13 (d, J = 2.0 Hz, 1H), 6.38-6.38 (d, J

= 2.0 Hz, 1H), 6.76 (s, 1H), 6.94-6.96 (d, J = 12.0 Hz, 2H), 7.82-7.84 (d, J = 12.0 Hz,
2H). 13C NMR (100 MHz, CDCl3) δ 180.67, 168.81, 168.71, 160.56, 159.31, 146.78,
132.89, 125.31, 114.34, 111.03, 105.45, 93.92, 89.14, 56.21, 56.10, 55.37. ESI-HRMS
calculated for C18H17O5 [M-HF+H]+: 313.1076, found: 313.1068.
(Z)-2-fluoro-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-e
n-1-one (29): yellowish powder, yield 71 %. 1H NMR (600 MHz, CDCl3): δ (ppm) =
3.87 (s, 3H), 3.90 (s, 3H), 3.95 (s, 3H), 6.14-6.14 (d, J = 3.0 Hz, 1H), 6.38-6.38 (d, J
= 3.0 Hz, 1H), 6.74 (s, 1H), 6.91-6.93 (dd, J = 6.0, 6.0 Hz, 1H), 7.32-7.36 (t, J = 6.0,
13
9.0 Hz, 1H), 7.44-7.45 (d, J = 9.0 Hz, 2H). C NMR (100 MHz, CDCl3) δ 180.68,
169.07, 169.03, 159.68, 159.42, 147.97, 133.79, 129.72, 123.87, 116.20, 115.09,
110.66, 105.17, 94.09, 89.26, 56.24, 56.16, 55.33. ESI-HRMS calculated for
C18H17O5 [M-HF+H]+: 313.1076, found: 313.1069.
(Z)-2-fluoro-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-phenylprop-2-en-1-one (30):
1
yellowish powder, yield 75 %. H NMR (300 MHz, CDCl3): δ (ppm) = 3.92 (s, 3H),
3.96 (s, 3H), 6.14-6.15 (d, J = 6.0 Hz, 1H), 6.40-6.40 (d, J = 6.0 Hz, 1H), 6.78 (s, 1H),
7.36-7.38 (t, J = 12.0 Hz, 1H), 7.42-7.44 (t, J = 6.0, 6.0 Hz, 2H), 7.86-7.88 (d, J =
12.0 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 180.73, 169.10, 169.00, 159.44, 147.88,
132.59, 131.12, 129.33, 128.79, 110.79, 105.24, 94.07, 89.25, 56.25, 56.15.
ESI-HRMS calculated for C17H15O4 [M-HF+H]+: 283.0970, found: 283.0963.
(Z)-2-fluoro-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(4-hydroxyphenyl)prop-2-e
n-1-one (31): yellowish powder, yield 49 %. 1H NMR (600 MHz, DMSO-d6): δ (ppm)
= 3.89 (s, 3H), 3.91 (s, 3H), 6.33-6.33 (d, J = 2.0 Hz, 1H), 6.65 (s, 1H), 6.68-6.68 (d,
13
J = 2.0 Hz, 1H), 6.86-6.88 (d, J = 12.0 Hz, 2H), 7.78-6.80 (d, J = 12.0 Hz, 2H). C
NMR (100 MHz, DMSO-d6) δ 178.85, 168.55, 167.89, 159.08, 158.74, 133.01,
123.05, 115.97, 110.40, 104.26, 94.23, 89.73, 56.42, 56.06. ESI-HRMS calculated for
C17H15O5 [M-HF+H]+: 299.0920, found: 299.0910.
(Z)-2-fluoro-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-p-tolylprop-2-en-1-one (32):
1
yellowish powder, yield 56 %. H NMR (600 MHz, CDCl3): δ (ppm) = 2.39 (s, 3H),
3.91 (s, 3H), 3.96 (s, 3H), 6.13-6.14 (d, J = 6.0 Hz, 1H), 6.39-6.40 (d, J = 6.0 Hz, 1H),
6.76 (s, 1H), 7.22-7.24 (d, J = 12.0 Hz, 2H), 7.75-7.77 (d, J = 12.0 Hz, 2H). 13C NMR

(100 MHz, CDCl3) δ 180.76, 168.99, 168.87, 159.37, 147.46, 139.78, 131.14, 129.78,
129.58, 111.07, 105.35, 93.99, 89.20, 56.23, 56.13, 21.60. ESI-HRMS calculated for
C18H17O4 [M-HF+H]+: 297.1127, found: 297.1120.
(Z)-2-fluoro-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)prop
-2-en-1-one (33): yellowish powder, yield 63 %. 1H NMR (600 MHz, CDCl3): δ (ppm)
= 3.92 (s, 3H), 3.94 (s, 3H), 3.96 (s, 3H), 3.97 (s, 3H), 6.15-6.15 (d, J = 6.0 Hz, 1H),
6.36-6.36 (d, J = 6.0 Hz, 1H), 6.75 (s, 1H), 6.92-6.93 (d, J = 9.0 Hz, 1H), 7.45-7.47 (d,
J = 6.0, 9.0 Hz, 2H).
(Z)-2-fluoro-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(2,5-dimethoxyphenyl)prop
-2-en-1-one (34): yellowish powder, yield 62 %. 1H NMR (600 MHz, CDCl3): δ (ppm)
= 3.86 (s, 6H), 3.90 (s, 3H), 3.95 (s, 3H), 6.13 (s, 1H), 6.36-6.36 (d, J = 2.0 Hz, 1H),
6.84-6.85 (d, J = 6.0 Hz, 1H), 6.88-6.90 (dd, J = 6.0, 3.0 Hz, 1H), 7.28 (s, 1H),
7.81-7.81 (d, J = 3.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 168.98, 166.04,
152.42, 149.28, 147.36, 143.38, 112.38, 99.50, 94.70, 90.03, 56.58, 56.15, 55.51.
ESI-HRMS calculated for C19H19O6 [M-HF+H]+: 343.1182, found: 343.1178.
(E)-1-(2’-acetoxy-4’,6’-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one
(35): 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one 94.3
mg (0.3 mmol, 1eq) was dissolved into 3 ml Ac2O and refluxed for 3 hours, after
work-up, the crude product was purified by Si-gel chromatography to afford 84.4 mg
1
yellowish oil, yield 79 %. H NMR (300 MHz, CDCl3): δ (ppm) = 2.18 (s, 3H), 3.80
(s, 3H), 3.82 (s, 3H), 3.84 (s, 3H), 6.30-6.30 (d, J = 3.0 Hz, 1H), 6.40-6.41 (d, J = 3.0
Hz, 1H), 6.91-6.95 (m, 2H), 7.00-7.04 (d, J = 12.0 Hz, 1H), 7.11-7.14 (d, J = 12.0 Hz,
1H), 7.28-7.31 (d, J = 9.0 Hz, 1H), 7.37-7.42 (d, J = 9.0 Hz, 1H). 13C NMR (100 MHz,
CDCl3) δ 191.68, 169.21, 162.15, 159.87, 159.19, 150.08, 144.25, 136.16, 129.88,
128.27, 121.12, 116.35, 115.80, 113.16, 100.26, 96.71, 56.06, 55.66, 55.30, 20.89.
ESI-HRMS calculated for C20H20O6Na [M+Na]+: 379.1158, found: 379.1156.
(E)-1-(2’-ethoxy-4’,6’-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one
(36): 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one 94.3
mg (0.3 mmol, 1eq) was dissolved into 10 ml acetone, K2CO3 (3 eq.) was added, the
mixture was stirred for a while, then bromoethane 46 mg (0.39 mmol, 1.3 eq) was

added. The mixture was refluxed until the starting material was consumed over. The
solvent was removed under reduced pressure, and the crude product was purified by
Si-gel chromatography （PE/EA=6/1）to afford 84 mg yellowish oil, yield 82 %. 1H
NMR (300 MHz, CDCl3): δ (ppm) = 1.28-1.31 (t, J = 6.0, 3.0 Hz, 3H), 3.84 (s, 3H),
3.85 (s, 3H), 3.92 (s, 3H), 5.97 (d, J = 6.0 Hz, 1H), 6.12 (d, J = 6.0 Hz, 1H), 6.94 (q, J
= 6.0, 6.0 Hz, 1H), 7.12 (s, 1H), 7.21 (d, J = 6.0 Hz, 1H), 7.32 (d, J = 6.0 Hz, 1H),
13
7.75 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 9.0 Hz, 1H). C NMR (100 MHz, CDCl3) δ
194.30, 162.40, 159.85, 158.91, 158.31, 143.76, 136.50, 129.80, 129.40, 121.06,
116.07, 113.04, 112.06, 91.57, 90.75, 64.38, 55.93, 55.46, 55.30, 14.67. ESI-HRMS
calculated for C20H22O5Na [M+Na]+: 365.1365, found: 365.1355.
1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3-methoxyphenyl)propan-1-one
(37):
1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one 94.3 mg
(0.3 mmol, 1eq) was dissolved into 5 ml MeOH, catalytic amount PtO2 was added.
The air was exchanged by H2 for three times, the mixture was stirred for 24 hours at
room temperature. The solvent was removed and the crude product was purified by
Si-gel column chromatography (PE/EA=8/1) to afford desired compound, white
powder, 53 mg, yield 56 %. 1H NMR (300 MHz, CDCl3): δ (ppm) = 2.97-3.02 (t, J =
9.0 Hz, 2H), 3.32-3.37 (t, J = 9.0 Hz, 2H), 3.84 (s, 3H), 3.85 (s, 3H), 3.92 (s, 3H),
5.94 (d, J = 3.0 Hz, 1H), 6.10 (d, J = 3.0 Hz, 1H), 6.77-7.22 (m, 3H), 7.22-7.28 (m,
1H), 14.07 (s, 1H).
5,7-dimethoxy-2-(3-methoxyphenyl)chroman-4-one
(38):
1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one 94.3 mg
(0.3 mmol, 1eq) was dissolved into 5 ml MeOH, followed by the addition of 3 ml 5N
aq. HCl solution, the mixture was refluxed for overnight, after the starting material
was consumed completely and cooled to room temperature, 20 ml water was added.
Extracted with ethyl acetate for three times, the organic layer was combined and dried,
the solvent was removed under reduced pressure. The residue was purified by Si-gel
chromatography (PE/EA=6/1) to afford desired compound, 59 mg, white solid, yield
62 %. 1H NMR (300 MHz, CDCl3): δ (ppm) = 2.73 (dd, J = 3.0, 3.0 Hz, 1H), 2.94 (dd,
J = 12.0, 12.0 Hz, 1H), 3.75 (s, 3H), 3.76 (s, 3H), 3.82 (s, 3H), 5.28 (dd, J = 3.0, 3.0

Hz, 1H), 6.02 (d, J = 3.0 Hz, 1H), 6.09 (d, J = 3.0 Hz, 1H), 6.83 (t, 1H), 7.19 (s, 2H),
7.23-7.29 (m, 1H). The analytical data were nicely consistent with related
reference[62].
(2E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one
oxime
(39):
1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one 94.3 mg
(0.3 mmol, 1eq) was dissolved into followed by the addition of hydroxylamine
hydrochloride 41.7 mg (0.6 mmol, 2eq) and piperidine (3.0 eq). The mixture was
stirred for 12 hours. After the starting material was consumed completely, the solvent
was removed. The residue was purified by Si-gel column chromatography
(PE/EA=6/1) to afford desired compound, 41.5 mg, white solid, yield 42 %. 1H NMR
(300 MHz, DMSO-d6): δ (ppm) = 3.64 (s, 3H), 3.74 (s, 3H), 3.75 (s, 3H), 6.10-6.13 (d,
J = 3.0, 6.0 Hz, 2H), 6.26-6.31 (d, J = 15.0 Hz, 1H), 6.83-6.86 (q, J = 3.0, 3.0 Hz, 1H),
6.95 (s, 1H), 7.01-7.04 (d, J = 9.0 Hz, 1H), 7.21-7.27 (t, J = 9.0, 9.0 Hz, 1H),
7.55-7.60 (d, J = 15.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 162.16, 159.80,
156.81, 138.32, 137.67, 129.64, 120.35, 116.83, 115.06, 112.45, 111.87, 102.40, 99.98,
93.54, 91.91, 55.73, 55.43, 55.31. ESI-HRMS calculated for C18H20NO5 [M+H]+:
330.1263, found: 330.1339.
(E)-1-(2’-hydroxy-4’-methoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (40):
yellowish solid, yield 78 %. 1H NMR (300 MHz, CDCl3): δ (ppm) = 3.83-3.86 (s, 6H),
6.47-6.50 (t, J = 3.0, 6.0 Hz, 2H), 6.93-6.96 (d, J = 9.0 Hz, 2H), 7.44-7.49 (d, J = 15.0
Hz, 1H), 7.60-7.63 (d, J = 9.0 Hz, 2H), 7.81-7.89 (t, J = 9.0, 15.0 Hz, 2H), 13.54 (s,
1H). The analytical data were nicely consistent with related reference[63].
(E)-1-(2’-hydroxy-4’-methoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one (41):
yellowish solid, yield 79 %. 1H NMR (300 MHz, CDCl3): δ (ppm) = 3.85-3.86 (s, 6H),
6.48-6.51 (t, J = 6.0, 3.0 Hz, 2H), 6.96-6.99 (m, 1H), 7.15-7.16 (d, J = 3.0 Hz, 1H),
7.32-7.37 (t, J = 9.0, 6.0 Hz, 1H), 7.53-7.59 (d, J = 18.0 Hz, 1H), 7.82-7.87 (t, J = 6.0,
9.0 Hz, 2H), 13.41 (s, 1H). The analytical data were nicely consistent with related
reference[64].
(E)-1-(2’-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (42): yellowish

solid, yield 81 %. 1H NMR (300 MHz, CDCl3): δ (ppm) = 3.96 (s, 3H), 6.91-7.04 (m,
4H), 7.48-7.84 (m, 4H), 7.88-7.93 (m, J = 6.0, 6.0 Hz, 2H), 12.93 (s, 1H). The
analytical data were nicely consistent with related reference[65].
4-(3-bromopropoxy)-3-methoxybenzaldehyde
(45):
4-hydroxy-3-methoxybenzaldehyde (1.52 g, 10.0 mmol, 1.0 eq) was dissolved into 20
ml MeCN, followed by the addition of K2CO3 (2.07 g, 15.0 mmol, 1.5 eq). After
stirred for a while, 1,3-dibromopropane (6.06 g, 30.0 mmol, 3.0 eq) was added into
o
the solution and refluxed at 85 C for about 4 hours. When the starting material was
consumed over, the solution was cooled to r.t. and the diluted with 120 ml water. The
mixture was extracted with EA (40 ml) for three times, the organic layer was
combined and dried with Na2SO4, filtered and the solvent was removed to give oil
residue. The residue was purified by Si-gel column chromatography to afford
compound 45 as white solid 2.46 g, yield 90.0%. 1H NMR (400 MHz, CDCl3) δ (ppm)
= 2.41-2.44 (m, 2H), 3.63-3.66 (t, J = 4.0, 8.0 Hz, 2H), 3.93 (s, 3H), 4.24-4.27 (t, J =
8.0, 4.0 Hz, 2H), 7.00-7.02 (d, J = 8.0 Hz, 1H), 7.42 (d, 1H), 7.44-7.47 (dd, J = 4.0,
8.0 Hz, 1H), 9.86 (s, 1H).
3-methoxy-4-(3-(pyrrolidin-1-yl)propoxy)benzaldehyde (46): compound 45
(546.24 mg, 2.0 mmol, 1.0 eq) was added into 10 ml MeCN, followed by the addition
of K2CO3 (414.63 mg, 3.0 mmol, 1.5 eq) and pyrrolidine (284.48 mg, 4.0 mmol, 2.0
eq). The mixture was refluxed at 85 oC for about 5 hours until the starting material 45
was consumed over. The solvent was removed under reduced pressure, and the
residue was purified by Si-gel column chromatography (DCM/MeOH = 15/1) to
1
afford compound 46 as pale white oil 442.4 mg, yield 84.0%. H NMR (400 MHz,
CDCl3) δ (ppm) = 1.80 (s, 4H), 2.10-2.15 (m, 2H), 2.57 (t, 4H), 2.66-2.69 (t, J = 8.0,
4.0 Hz, 2H), 3.92 (s, 3H), 4.17-4.21 (t, J = 8.0, 8.0 Hz, 2H), 7.00-7.02 (d, J = 8.0 Hz,
1H), 7.40 (d, 1H), 7.42-7.45 (dd, J = 4.0, 4.0 Hz, 1H), 9.84 (s, 1H).
3-methoxy-4-(3-(piperidin-1-yl)propoxy)benzaldehyde (47): compound 45 (546.24
mg, 2.0 mmol, 1.0 eq) was added into 10 ml MeCN, followed by the addition of
K2CO3 (414.63 mg, 3.0 mmol, 1.5 eq) and piperidine (340.44 mg, 4.0 mmol, 2.0 eq).
The mixture was refluxed at 85 oC for about 5 hours until the starting material 45 was

consumed over. The solvent was removed under reduced pressure, and the residue
was purified by Si-gel column chromatography (DCM/MeOH = 15/1) to afford
compound 47 as pale white oil 504.8 mg, yield 91.0%. 1H NMR (400 MHz, CDCl3) δ
(ppm) = 1.46-1.47 (brd, J = 4.0 Hz, 2H), 1.60-1.65 (m, 4H), 2.07-2.14 (m, 2H), 2.46
(br, 4H), 2.53-2.56 (t, J = 4.0, 8.0 Hz, 2H), 3.92 (s, 3H), 4.16-4.20 (t, J = 8.0, 8.0 Hz,
2H), 7.00-7.02 (d, J = 8.0 Hz, 1H), 7.40-7.41 (d, J = 4.0 Hz, 1H), 7.43-7.45 (dd, 1H),
9.85 (s, 1H).
3-methoxy-4-(3-(4-methylpiperazin-1-yl)propoxy)benzaldehyde (48): compound
45 (546.24 mg, 2.0 mmol, 1.0 eq) was added into 10 ml MeCN, followed by the
addition of K2CO3 (414.63 mg, 3.0 mmol, 1.5 eq) and N-methyl piperazine (400.64
o
mg, 4.0 mmol, 2.0 eq). The mixture was refluxed at 85 C for about 5 hours until the
starting material 45 was consumed over. The solvent was removed under reduced
pressure, and the residue was purified by Si-gel column chromatography
(DCM/MeOH = 15/1) to afford compound 48 as pale yellowish solid 514.5 mg, yield
88.0%.1H NMR (400 MHz, CDCl3) δ (ppm) = 2.05-2.08 (m, 2H), 2.22 (s, 1H), 2.29 (s,
3H), 2.46-2.56 (m, 9H), 3.92 (s, 3H), 4.16-4.20 (t, J = 8.0, 8.0 Hz, 2H), 7.00-7.02 (d,
J = 8.0 Hz, 1H), 7.41 (d, 1H), 7.43-7.45 (dd, 1H), 9.85 (s, 1H).
3-methoxy-4-(3-morpholinopropoxy)benzaldehyde (49): compound 45 (546.24 mg,
2.0 mmol, 1.0 eq) was added into 10 ml MeCN, followed by the addition of K2CO3
(414.63 mg, 3.0 mmol, 1.5 eq) and morpholine (348.48 mg, 4.0 mmol, 2.0 eq). The
o
mixture was refluxed at 85 C for about 5 hours until the starting material 45 was
consumed over. The solvent was removed under reduced pressure, and the residue
was purified by Si-gel column chromatography (DCM/MeOH = 15/1) to afford
compound 49 as pale white oil 519.6 mg, yield 93.0%. 1H NMR (400 MHz, CDCl3) δ
(ppm) = 2.03-2.10 (m, 2H), 2.47 (br., 2H), 2.52-2.56 (t, J = 8.0, 8.0 Hz, 2H),
3.70-3.73 (t, J = 4.0, 8.0 Hz, 4H), 3.92 (s, 3H), 4.17-4.21 (t, J = 8.0, 8.0 Hz, 2H),
7.00-7.02 (d, J = 8.0 Hz, 1H), 7.40-7.41 (d, J = 4.0 Hz, 1H), 7.43-7.45 (dd, 1H), 9.85
(s, 1H).
(E)-1-(2’-hydroxy-4’,6’-dimethoxyphenyl)-3-(3-methoxy-4-(3-(pyrrolidin-1-yl)pr
1
opoxy)phenyl)prop-2-en-1-one (50): yellowish solid, yield 63 %. H NMR (600