Synthesis and biological evaluation of chalcone, dihydrochalcone, and 1,3-diarylpropane analogs as anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2017.02.038

Twenty-one chalcones were prepared via aldol condensation and subsequent reduction of these compound led to the corresponding dihydrochalcone and 1,3-diphenylpropane derivatives. The synthetic products were examined for their effects on NO inhibition in L

Full text of DOI:10.1016/j.bmcl.2017.02.038

Accepted Manuscript
Synthesis and biological evaluation of chalcone, dihydrochalcone, and 1,3-di-
arylpropane analogs as anti-inflammatory agents
Mopur Vijaya Bhaskar Reddy, Hsin-Yi Hung, Ping-Chung Kuo, Guan-Jhong
Huang, Yu-Yi Chan, Shiow-Chyn Huang, Shwu-Jen Wu, Susan L. Morris-
Natschke, Kuo-Hsiung Lee, Tian-Shung Wu
PII:
S0960-894X(17)30171-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.038
BMCL 24710
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
12 January 2017
14 February 2017
16 February 2017
Please cite this article as: Vijaya Bhaskar Reddy, M., Hung, H-Y., Kuo, P-C., Huang, G-J., Chan, Y-Y., Huang, S-
C., Wu, S-J., Morris-Natschke, S.L., Lee, K-H., Wu, T-S., Synthesis and biological evaluation of chalcone,
dihydrochalcone, and 1,3-diarylpropane analogs as anti-inflammatory agents, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.038
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Synthesis and biological evaluation of chalcone, dihydrochalcone, and
1,3-diarylpropane analogs as anti-inflammatory agents
Mopur Vijaya Bhaskar Reddy a,#, Hsin-Yi Hung b,#, Ping-Chung Kuo b,#, Guan-Jhong
Huang c, Yu-Yi Chan d, Shiow-Chyn Huang e, Shwu-Jen Wu f, Susan L. Morris-Natschke g,
Kuo-Hsiung Lee g,h,*, Tian-Shung Wu b,i,*
a Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan,
b School of Pharmacy, National Cheng Kung University Hospital, College of Medicine, National
Cheng Kung University, Tainan 701, Taiwan
c
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of
Pharmacy, China Medical University, Taichung 404, Taiwan
d Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan
710, Taiwan
e Graduate Institute of Pharmaceutical Science, Chia-Nan University of Pharmacy and Science,
Tainan 717, Taiwan
f Department of Medical Technology, Chung Hua University of Medical Technology, Tainan 717,
Taiwan
g Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of
North Carolina, Chapel Hill, NC 27599, United States
1

h Chinese Medicinal Research and Development Center, China Medical University Hospital,
China Medical University, Taichung 404, Taiwan
i Department of Pharmacy, College of Pharmacy and Health Care, Tajen University, Pingtung 907,
Taiwan
To whom correspondence should be addressed. Tian-Shung Wu: Tel: 886-6-2747538. Fax:
886-2-2740552. E-mail: tswu@mail.ncku.edu.tw. Kuo-Hsiung Lee: Tel: +919-962-0066. Fax:
+919-966-3893. Email: khlee@unc.edu
# Authors contributed equally to this work
2

Abstract
Twenty-one chalcones were prepared via aldol condensation and subsequent reduction of
these compound led to the corresponding dihydrochalcone and 1,3-diphenylpropane derivatives.
The synthetic products were examined for their effects on NO inhibition in LPS-activated mouse
peritoneal macrophages. Among the tested compounds, a 1,3-diarylpropane analog,
2-(3-(3,4-dimethoxyphenyl)propyl)-5-methoxyphenol (3p), displayed the most significant
inhibitory effects against NO production. To investigate the mechanism of action, the effects of
3p on iNOS and COX-2 protein expression were studied by immunoblot. The results concluded
that 3p is capable of inhibiting iNOS expression in LPS-induced RAW264.7 cells via attenuation
of NF-κB signaling by ERK, p38, and JNK.
Keywords:
Chalcones
Dihydrochalcones
1,3-Diarylpropane analogs
Anti-inflammatory agents
3

Flavonoids are a ubiquitous group of polyphenolic substances concentrated in the seeds,
fruit skin or peel, bark, and flowers of most plants. Numerous plant medicines contain flavonoids,
which have been reported by many authors as having antibacterial, anti-inflammatory,
anti-allergic, antimutagenic, antiviral, antineoplastic, and vasodilatory actions. Many studies
have shown that some flavonoids are potent antioxidants that are capable of scavenging hydroxyl
·-
radicals (.OH), superoxide anions (O2 ), lipid peroxy radicals, and hydrogen peroxide (H2O2).
These free radicals have been implicated several disease processes, including asthma,1,2 cancer,3
cardiovascular disease,4,5 cataracts,6,7 diabetes,8,9 gastrointestinal inflammatory diseases,10,11 liver
disease,12 mascular degeneration,13,14 periodontal disease,15 and other inflammatory processes. In
the flavonoid family, chalcones and dihydrochalcones belong to a major class of bicyclic
compounds, which are precursors for flavonoid biosynthesis in plants. The two aromatic rings in
these compounds are linked by a three carbon bridge, specifically, propenone in chalcones and
propanone in dihydrochalcones. Compounds of both types exert multiple biological activities,
including anti-inflammatory, antioxidant and anticancer properties.16-21 In addition, viscolin, a
1,3-diarylpropane, displayed potent and selective inhibition on the superoxide anion generation
activated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) combined with cytochalasin B in
human neutrophils, and also exhibited free radical scavenging effects in
a
1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay. Both actions could be mediated by viscolin’s
4

anti-inflammatory activity.22 Anti-inflammatory and antioxidant therapies are also
comprehensive pharmacological approaches in the treatment of inflammatory related
disorders.23,24
In the present study, we synthesized three corresponding series of chalcone,
dihydrochalcone, and 1,3-diphenylpropane derivatives (Fig. 1, details in Supplementary Data)
and examined the compounds for in vitro anti-inflammatory activity using a lipopolysaccharide
(LPS)-stimulated RAW264.7 cellular assay. We also evaluated the effect of a selected compound
(3p) on nuclear factor kappa B (NF-κB) expression and mitogen-activated protein kinase
(MAPK) signaling pathways to better understand the molecular mechanism.
5

OH
R4
R1
OH
R4
R1
OH
R4
R1
R3
R2
R3
R2
R3
R2
a
b
O
O
3-series
2-series
1-series
1a, 2a, 3a). R1 = R2 = R4 = H, R3 = OMe
1b, 2b, 3b). R1 = R2 = OMe, R3 = R4 = H
1c, 2c, 3c). R1 = R3 = OMe, R2 = R4 = H
1d, 2d, 3d). R1 = R4 = OMe, R2 = R3 = H
1e, 2e, 3e). R2 = R3 = OMe, R1 = R4 = H
1f, 2f, 3f). R1 = R4 = H, R2 = OMe, R3 = OH
R4
R4
R4
R7
R6
R3
R2
R7
R6
R3
R2
R7
R6
R3
R2
a
c
R5
O
R1
R5
O
R1
R5
R1
3-series
2-series
1-series
1g, 2g, 3g). R1 = R4 = R5 = R6 = H, R7 = OH, R2 = R3 = -OCH2O-
1h, 2h, 3h). R1 = R2 = R4 = R5 = H, R6 = R3 = OMe, R7 = OH
1i, 2i, 3i).
1j, 2j, 3j).
R1 = R4 = R5 = H, R6 = OMe, R7 = OH, R2 = R3 = -OCH2O-
R1 = R2 = R6 = OMe, R7 = OH, R3 = R4 = R5 = H
1k, 2k, 3k). R1 = R2 = R4 = R5 = H, R3 = R6 = R7 = OMe
1l, 2l, 3l). R1 = R4 = R5 = H, R2 = R3 = R6 = R7 = OMe
1m, 2m, 3m). R1 = R4 = R5 = H, R2 = OH, R3 = R6 = R7 = OMe
1n, 2n, 3n). R1 = R4 = R6 = H, R5 = R7 = OMe, R2 = R3 = -OCH2O-
1o, 2o, 3o). R1 = R4 = R6 = H, R5 = OH, R7 = OMe, R2 = R3 = -OCH2O-
1p, 2p, 3p). R1 = R4 = R6 = H, R5 = OH, R2 = R3 = R7 = OMe
1q, 2q, 3q). R1 = R2 = R4 = R6 = H, R5 = OH, R3 = R7 = OMe
1r, 2r, 3r).
1s, 2s, 3s). R3 = R4 = R6 = H, R1 = R2 = R7 = OMe, R5 = OH
1t, 2t, 3t). R2 = R4 = R6 = H, R1 = R3 = R7 = OMe, R5 = OH
1u, 2u, 3u). R2 = R3 = R6 = H, R1 = R4 = R7= OMe, R5 = OH
R4 = R6 = H, R1 = R2 = R3 = R7 = OMe, R5 = OH
Fig. 1. The chemical structures of the chalcone, dihydrochalcone, and 1,3-diarylpropane analogs.
Reagents and conditions: (a) Pd/C, H2, EtOAc; (b) Ni-Al alloy (50:50), H2O, reflux at 110-120
C for 8-12 h; (c) TFA, (C2H5)3SiH, reflux at 50-55 C for 12 h.
6

Chalcones 1a-1u were prepared by an aldol condensation of substituted acetophenones
and aldehydes in equimolar quantities with KOH as base at room temperature (Fig. 1).25 The
stereochemistry of the olefinic carbon-carbon bond of each chalcone was established based on
the appropriate 1H NMR coupling constant. Chalcones 1a-1u then underwent hydrogenation in a
H2 atmosphere with 10% Pd-C as catalyst and EtOAc as solvent for 24‒48 h to obtain
dihydrochalcones 2a-2u, respectively, in good yields (Fig. 1).26 To prepare the analogous
1,3-diarylpropanes 3a-3u, we needed to reduce the carbonyl groups in 2a-2u to methylene units.
To the best of our knowledge, no literature exists on this specific reduction in dihydrochalcones.
Also, drawbacks exist to the two common methods used for this conversion: the Wolf-Kishner
reaction,27 which cannot be applied to base sensitive substrates, and the Clemmensen reaction,
which is not suitable for acid sensitive precursors, often requires harsh conditions and long
reaction times, and suffers from poor yields.28, 29 So herein, we tested two methods for the
preparation of 1,3-diphenylpropanes from dihydrochacones. In method A, dihydrochalcones
2a-2f and Ni-Al alloy (50:50)30 were combined in H2O at room temperature and the reaction
mixture was stirred vigorously under reflux for 8‒12 h giving 1,3-diphenylpropanes 3a-3f,
respectively, in good yields. However, when we applied the same procedure to compounds 2g-2u,
which have oxygenated substituents at C-4, C-2,4, or C-3,4 in ring A, no reaction was observed
in most cases, likely due to conjugation and chelation effects. Subsequently, in method B,
7

dihydrochalcones 2g-2u were stirred with trifluroacetic acid (TFA) and triethylsilyl hydride
[(C2H5)3SiH]31 at 0 C for 30 min and then at 50‒55 C for 12 h, to provide
1,3-diphenylpropanes 3g-3u, respectively, in good yields (Fig. 1).
Cell viability and effect of 3p on LPS-induced NO production in macrophages
We evaluated the effects of the synthesized chalcones, dihydrochalcones, and
1,3-diphenylpropane derivatives at four concentrations (1.25, 2.5, 5, and 10 μg/mL) on
RAW264.7 cell viability in a MTT assay as well as on NO production by measuring nitrite levels
based on the Griess reaction in LPS-activated mouse peritoneal macrophages.18 NO production
was significantly decreased in a dose-dependent manner by the treatment with various
compounds from all three series. Compounds 1h, 1k, 2e‒2i, 2k, 3a, 3g, 3i‒3m, 3p, and 3r
exhibited IC50 values in a range of 2.0 to 9.8 μg/mL (Tables 1‒3, Supplementary Material),
without a significant influence on cell viability at that concentration. Thus, the NO inhibitory
effects of these compounds were probably not due to cytotoxic effects. Some compounds did
affect cell viability, especially at the highest dose level. For example, although 1d with a
2,5-dimethoxyphenyl ring had a low NO inhibitory IC50 value (2.42 μg/mL), it also reduced cell
viability at that concentration (28% reduction at 2.5 μg/mL). The NO inhibitory ability did not
follow similar relationships among the 1-, 2-, 3-compounds, which may suggest different binding
8

modes among these three series. Overall, 1,3-diarylpropane analog 3p exhibited the best
anti-inflammatory activity among the tested compounds. Other biological studies on this
compound have not been reported, and the mechanisms underlying the anti-inflammatory
activity have not yet been elucidated. Therefore, in the present study, we investigated possible
molecular mechanisms of 3p using LPS-stimulated RAW 264.7 cells in vitro.
Inhibition of LPS-induced iNOS and COX-2 protein by 3p
To investigate whether the inhibition of NO production was due to decreased iNOS and
COX-2 protein levels, the effect of 3p on iNOS and COX-2 protein expression was studied by
using an immunoblot. Incubation with 3p (2.5, 5, and 10 μg/mL) in the presence of LPS (100
ng/mL) for 24 h inhibited iNOS protein expression in mouse macrophage RAW264.7 cells in a
dose-dependent manner (Fig. 2A). The detection of β-actin was also performed in the same blot
as an internal control.
9

A.
B.
C.
10

Fig. 2. Inhibition of iNOS, COX-2 (A), MAPK (ERK, JNK, and p38) (B), and NF-κB (C)
protein expression by 3p in LPS-stimulated RAW264.7 cells. Cells were incubated for 24 h (2A)
or 1 h (2B, 2C) with 100 ng/mL of LPS in the absence or presence of 3p (0, 2.5, 5, and 10
g/mL). Compound 3p was added 1 h before the incubation with LPS. Lysed cells were then
prepared and subjected to Western blotting by using an antibody specific for iNOS, COX-2,
MAPK, or NF-κB. β-Actin was used as an internal control.
Effects of 3p on the LPS-stimulated activation of MAPKs
MAPKs play critical roles in the regulation of cell growth and differentiation, and control
cellular responses to cytokines and stresses. In particular, ERK, p38, and JNK are important for
the activation of NF-κB.20,21 To explore whether the inhibition of NF-κB activation by 3p is
mediated through the MAPK pathway, MAPK phosphorylation was examined by Western blot
in RAW 264.7 cells pretreated with 3p and then with LPS. As shown in Fig. 2B, 3p suppressed
the LPS-induced activation of ERK, JNK, and p38 MAPKs. However, the expression of
non-phosphorylated ERK, JNK, and P38 MAPKs was unaffected by LPS or LPS plus 3p. These
results suggest that phosphorylation of MAPKs may be involved in the inhibitory effect of 3p on
11

LPS-stimulated NF-κB binding in RAW 264.7 cells.
Inhibition of LPS-induced NF-κB proteins by 3p
The effect of NF-κB expression by 3p in the presence of LPS for 1 h was assessed by
Western blotting. The intensity of protein bands showed an average of 79.2% increase of NF-κB
protein after treatment with 3p at 2.5, 5, and 10 μg/mL compared with LPS alone (Fig. 2C).
Therefore, we concluded that 3p is capable of inhibiting iNOS expression in LPS induced
RAW264.7 cells via attenuation of NF-κB signaling by ERK, p38, and JNK.
In summary, the synthesized chalcones, dihydrochalcones and 1,3-diarylpropanes were
studied for their anti-inflammatory activity in vitro. This study suggested that some
dihydrochalcones and 1,3-diarylpropanes, especially compound 3p, may have the potential to be
developed as anti-inflammatory agents. However, further studies are necessary to more closely
examine the underlying molecular mechanisms and verify direct targets at the transcriptional or
post-transcriptional level in vitro.
Acknowledgments
This work was supported by a grant (NSC 96-2628-M-006-002) from the National Science
12

Council, Taiwan, and a grant (OUA 95-3-2-021) from National Cheng Kung University, Tainan,
Taiwan, awarded to T. S. Wu. This study was also supported in part by the Taiwan Department of
Health Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004).
Supplementary data
Supplementary data [Experimental procedures, NO inhibitory effects of synthesized
chalcones (Table 1), dihydrochalcones (Table 2), and 1,3-diarylpropanes (Table 3)] can be found,
in the online version, at
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