Halo-substituted chalcones and azachalcones-inhibited, lipopolysaccharited-stimulated, pro-inflammatory responses through the TLR4-mediated pathway

Article abstract of DOI:10.3390/molecules23030597

A series of B-ring, halo-substituted chalcones and azachalcones were synthesized to evaluate and compare their anti-inflammatory activity. Mouse BALB/c macrophage RAW 264.7 were pre-treated with 10 μg/mL of each compound for one hour before induction of i

Full text of DOI:10.3390/molecules23030597

molecules
Article
Halo-Substituted Chalcones and Azachalcones
Inhibited Lipopolysaccharited-Stimulated
Pro-Inflammatory Responses through the
TLR4-Mediated Pathway
Tzenge-Lien Shih 1, Ming-Hwa Liu 2, Chia-Wai Li 1 and Chia-Feng Kuo 2,
*
ID
1
Department of Chemistry, Tamkang University, Tamsui Dist., New Taipei City 251, Taiwan;
tlshih@mail.tku.edu.tw (T.-L.S.); 602180076@s02.tku.edu.tw (C.-W.L.)
Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University,
Zhongshan Dist., Taipei 104, Taiwan; a0141011@g2.usc.edu.tw
2
*
Correspondence: drkuo@g2.usc.edu.tw; Tel.: +886-2-2538-1111 (ext. 6214); Fax: +886-2-2533-4789
Received: 4 February 2018; Accepted: 4 March 2018; Published: 7 March 2018
Abstract: A series of B-ring, halo-substituted chalcones and azachalcones were synthesized to
evaluate and compare their anti-inflammatory activity. Mouse BALB/c macrophage RAW 264.7 were
pre-treated with 10
lipopolysaccharide (1
of pro-inflammatory factors toll-like receptor 4 (TLR4), I
(IL-1 ), IL-6, tumor necrosis factor (TNF- ), and cyclooxygenase 2 (COX-2). The present results
µ
g/mL of each compound for one hour before induction of inflammation by
g/mL) for 6 h. Some halo-chalcones and -azachalcones suppressed expression
B- , transcription factor p65, interleukine
µ
κ
α
1
β
β
α
α
showed that the synthetic halo-azachalcones exhibited more significant inhibition than halo-chalcones.
Therefore, the nitrogen atom in this series of azachalcones must play a more crucial role than the
corresponding C-2 hydroxyl group of chalcones in biological activity. Our findings will lay the
background for the future development of anti-inflammatory nutraceuticals.
Keywords: azachalcones; chalcones; anti-inflammation; toll-like receptor 4
1. Introduction
Inflammation is an immune response to injury and infection. Inflammation is considered to
be beneficial when it is short term and under control; however, chronic production of radicals by
inflammation causes oxidative damages and leads to chronic diseases [1–5]. Natural products that
possess anti-inflammatory properties are valuable phytomedicine.
Chalcones belong to the flavonoid family. They are widely distributed in natural products and
exhibit a variety of biological activities, including anti-oxidation, anti-inflammation, chemoprevention,
cardioprotection, antidiabetics, and neuroprotection [
have been designed and synthesized [15–21].
6–14]. Novel chalcones with biological activities
The skeleton of chalcones possesses a 1,3-diphenyl-2E-propene-1-one framework (Figure 1).
The hydroxy and methoxy groups are the most common substituted groups on either or both rings
in chalcones that show good antitubulin and cytotoxic activity [22]. Among them, the nearby C20
hydroxyl moiety in ring A contributed hydrogen bonding to the carbonyl group, leading to the
stabilization of the planar structure [23]. The most common strategy for the synthesis of chalcones is
Claisen-Schmidt condensation [24,25].
Molecules 2018, 23, 597; doi:10.3390/molecules23030597
www.mdpi.com/journal/molecules

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Figure 1. Structures of chalcones and azachalcones.
Azachalcone is another class of the chalcone family in which the carbons of either rings A or
B or both were replaced with a nitrogen atom. Because of their structural similarity, azachalcones
were synthesized with the same strategy as for chalcones, in which the substituted acetophenone was
replaced with 2-acetyl pyridine [26,27]. Studies have shown that azachalcones possess anti-bacterial,
anti-inflammatory, and anti-cancer properties [28
synthesis of pyrazolines and pyrazoles [32].
–31]. Azachalcones also served as precursors for
The halo-substituted chalcones and azachalcones are not naturally occurring compounds [33].
The halogen substitution in ring B of chalcones alters the electron distribution owing to its high
electronegativity and dipole moment, which can influence the biological activity. The aim of this study
is to synthesize novel, monohalogen-substituted chalcones and azachalcones in various positions in
ring B and evaluate their anti-inflammatory activity. To the best of our knowledge, this article is the first
report in a systematic study of the biological activities of monohalogen-substituted chalcones (7a
azachalcones (9a ) together (vide infra). The anti-inflammatory activities of synthetic chalcones and
azachalcones were also compared with those of synthetic, naturally occurring chalcones (Figure 2),
–i) and
–i
1–4
which were synthesized; their spectroscopic data were all in agreement with the reported values.
Figure 2. Natural chalcones 1–4.
2. Results and Discussion
2.1. Synthesis of Halo-Substituted Chalcones 7a–i and Azachalcones 9a–i
In order to optimize the reaction condition, we repeated the synthesis of a series of halo-substituted
chalcones 7g–i from 5 and halobenzaldehyde 6a–i by Claisen-Schmidt condensation (Figure 3).
The optimized yields were listed in Table 1.
Figure 3. Claisen-Schmidt condensation in synthesis of chalcones 7a–i.
Azachalcones 9a
recently [33]. The structures of synthetic chalcones 7a
The 1H and 13C NMR data for compounds 7a–i and 9a–i are available as supporting materials.
–
i
were synthesized from
8
and corresponding 6a
–
h
(Figure 4) as reported
–
i
and azachalcones 9a
–i are listed in Figure 5.

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Table 1. Optimization in synthesis of chalcones 7a–i.
Entry
Compound
Time (h)
Yield%
1
2
3
4
5
6
7
8
9
7a
7b
7c
7d
7e
7f
7g
7h
7i
14
9
15
8
13
14
13
14
15
92
75
82
76
67
73
85
78
73
Condition: 5 (1 equiv.), 6a–i (1.5 equiv.), KOH (3 equiv.), EtOH (0.2 M), and 0 to r.t.
Figure 4. Conditions of synthesis of azachalcones 9a–i.
Figure 5. Chemical structures of synthetic chalcones 7a–i and azachalcones 9a–i in this study.

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2.2. Biological Evaluation
2.2.1. Cytotoxic Effect of Halo-Substituted Chalcones (7a–i) and Azachalcones (9a–i)
To investigate the cytotoxicity of halo-substituted chalcones 7a
natural products
for 24 h. The viable cell numbers were assayed and compared to the control group. According to the
results in Table 2, 10 g/mL for each compound was chose to treat RAW 264.7 for the evaluation of
–
i
, azachalcones 9a
–i
, and synthetic
1–4, the RAW 264.7 cells were treated with various concentrations of each compound
µ
anti-inflammatory activities.
Table 2. Cell viability of RAW 264.7 cells after treated with various concentrations of halo-substituted
chalcones 7a–i, azachalcones 9a–i, or synthetic natural products 1–4 for 24 h.
Concentration (µg/mL)
Compound
0
10
25
50
100
114.27 ± 4.83 1
83.55 ± 12.07
110.86 ± 29.05
97.37 ± 20.37
83.42 ± 13.10
71.29 ± 8.12
80.47 ± 8.38
101.07 ± 8.63
87.00 ± 18.69
7a
7b
7c
7d
7e
7f
7g
7h
7i
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
99.76 ± 18.08
105.08 ± 14.37
115.31 ± 15.25
86.35 ± 18.47
69.68 ± 5.17
56.92 ± 4.88
75.60 ± 12.85
87.16 ± 9.03
64.16 ± 6.38
95.02 ± 21.09
90.34 ± 19.48
79.03 ± 6.30
71.78 ± 10.27
45.95 ± 6.08
30.60 ± 4.96
68.21 ± 7.45
60.71 ± 7.09
32.19 ± 3.32
80.32 ± 15.54
55.51 ± 4.21
65.77 ± 12.03
34.72 ± 4.15
17.24 ± 1.29
19.32 ± 2.89
42.19 ± 4.87
16.35 ± 0.56
24.23 ± 2.00
9a
9b
9c
9e
9f
9g
9h
9i
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
99.00 ± 3.19
123.61 ± 0.10
93.28 ± 9.09
90.39 ± 10.15
102.57 ± 16.87
86.56 ± 12.35
84.49 ± 8.66
79.14 ± 11.53
93.66 ± 3.87
112.57 ± 8.72
69.54 ± 23.80
85.06 ± 8.59
71.97 ± 8.07
89.25 ± 5.68
86.32 ± 8.10
77.95 ± 10.43
70.68 ± 2.17
82.18 ± 3.87
14.51 ± 2.05
63.56 ± 3.03
50.11 ± 9.90
34.25 ± 3.08
60.61 ± 25.05
67.49 ± 8.26
4.22 ± 0.47
3.92 ± 0.19
5.52 ± 0.37
8.60 ± 1.06
15.27 ± 1.64
3.3 ± 0.22
43.90 ± 18.91
33.68 ± 5.03
0
1
5
10
25
1
2
3
4
100.0
100.0
100.0
100.0
1
105.09 ± 12.45
99.80 ± 14.60
86.98 ± 6.15
90.63 ± 16.23
103.04 ± 5.41
102.52 ± 10.87
81.19 ± 8.43
75.05 ± 7.95
87.14 ± 14.13
64.09 ± 4.80
85.54 ± 12.25
23.64 ± 4.82
51.99 ± 6.98
34.01 ± 4.21
45.88 ± 6.48
103.44 ± 8.52
The results are presented as % of cell number of control group.
2.2.2. Effect of Halo-Substituted Chalcones (7a–i) and Azachalcones (9a–i) on mRNA and Protein
Expression of Pro-Inflammatory Factors
Inflammation is one of the pathologic processes that contribute to disorders. It involves
complex signal transduction pathways and the production of various pro-inflammatory factors.
Mammalian toll-like receptors (TLRs) are transmembrane receptors that recognize microbial infection
and trigger innate immune responses [34]. TLR4 is primarily expressed in macrophages. Binding of
lipopolysaccharide (LPS) to TRL4 initiates several signal transduction pathways, including NF-
NF- B is a heterodimer consisting of p65 and a p52 or p50. In cytoplasm, inactivated NF- B binds
with I B- . Phosphorylation of I B- results in the dissociation of NF- B from I B- , allowing the
translocation of heterodimer into the nucleus and binding to the promoters of pro-inflammatory
κB [35].
κ
κ
κ
α
κ
α
κ
κ α
genes, such as interleukine (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2
(Figure 6).

Molecules 2018, 23, 597
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Figure 6. TLR4-mediated NF-κB signaling pathway.
Pre-treatment with some synthetic chalcones and azachalcones ameliorated the expression of
TLR-4, IL-1 , IL-6, TNF- , and COX-2 induced by LPS (Figures 7 and 8). IL-1 , IL-6, and TNF-
are pro-inflammatory cytokines secreted by macrophages to initiate and regulate the progression
of inflammation [36]. IL-1 is the master inflammatory cytokine in the IL-1 family produced at the
early stages of the immune response. IL-1 plays a crucial role in recruitment of monocytes to the
inflammation site and interactions between immune cells and nerve cells [37]. Similar to IL-1 , IL-6
is an endogenous pyrogen that promotes fever and the production of acute phase proteins from
liver. IL-6 also involves in the recruitment of immune cells to inflammation site [38]. TNF- was
first described for its ability to induce necrosis of tumor cells. Binding of TNF- to receptors TNFR1
or TNFR2 signals NF- B activation. Transcriptional induction of TNF- by NF- B further amplifies
the signaling pathway [39 40]. COX catalyzes the conversion of fatty acids to prostaglandins and
thromboxanes. COX-1 is constitutively expressed in all cells, but the expression of COX-2 is induced by
pro-inflammatory cytokines [41]. Blocking of IL-1 , IL-6, TNF- , and COX-2 has become the targeting
therapy for the treatment of inflammatory diseases [42–45].
β
α
β
α
β
β
β
α
α
κ
α
κ
,
β
α

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100
(a)
TLR-4
a
b
80
60
40
c
d
d
20
0
def
de
defg
efgh
defg
efgh
efgh
efgh
fgh
fgh
fgh
fgh
7i 9a
fgh
gh
LPS 7a
gh
gh
gh
h
9g
-LPS
7b 7c
7e
7g
9f
9h 9i
1
2
7d
7f
7h
9b 9c 9e
3
4
500
IL-1β
(b)
a
400
300
200
100
0
bc
c
c
c
c
c
c
c
c
c
c
c
bc
c
bc
c
c
c
c
bc
1
b
2
bc
3
-LPS
7a 7b 7c 7d
7i
9g
4
LPS
7e 7f 7g 7h
9a 9b
9f
9c 9e
9h 9i
3000
2500
2000
1500
1000
(c)
IL-6
a
b
bc
bc
bc
c
d
500
0
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
7a 7b 7c
7g
7i 9a
9b 9c 9e
-LPS
9g 9h
1
3
LPS
7d 7e 7f
7h
4
9f
9i
2
Figure 7. mRNA expression of (
with synthetic or natural compounds (10
(1 g/mL) for 6 h. Values indicate means
= 0.05). LPS: lipopolysaccharide; 7a
halo-substituted azachalcones; 1–4 are synthetic natural products.
a
) TLR-4, (
b
) IL-1
β, and (c) IL-6. RAW 264.7 cells were pre-treated
µ
g/mL) for 1 h before induction of inflammation by LPS
SD. Bars with the same letters are not significantly different
are synthetic halo-substituted chalcones; 9a are synthetic
µ
±
(
α
–
i
–i

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20
(a)
a
18
16
14
12
10
8
TNF-α
b
bc
cde
6
bcd
cdef
defgh
efgh
defgh
defg
defgh
defgh
defgh
4
defgh
efgh
fghi
ghi
fghi
ghi
ghi
hi
ij
2
j
0
-LPS LPS 7a 7b 7c
7f 7g 7h
7d 7e
7i 9a
9g
3
9b 9c 9e
4
9h
9f
9i
1
2
35
(b)
COX-2
a
30
25
20
15
10
b
c
d
de
e
f
fg
fgh
fghij
fghi
fghij
fghij
ghij
5
0
hij
ghij
ghij
hij
ghij
ji
hij
ij
j
7b 7c
7g
7i 9a
9g
7a
9f
9h 9i
2
3
7d 7e 7f
7h
9b 9c 9e
1
4
-LPS LPS
Figure 8. mRNA expression of (
synthetic or natural compounds (10
for 6 h. Values indicate means
= 0.05). LPS: lipopolysaccharide; 7a
halo-substituted azachalcones; 1–4 are synthetic natural products.
a
) TNF-
α
and (
b
) COX-2. RAW 264.7 cells were pre-treated with
g/mL)
SD. Bars with the same letters are not significantly different
are synthetic halo-substituted chalcones; 9a are synthetic
µg/mL) for 1 h before induction of inflammation by LPS (1 µ
±
(
α
–
i
–i
The anti-inflammatory effects are different among the testing compounds; however,
halo-substituted azachalcones 9a showed more significant inhibition than halo-substituted chalcones
7a . Among the azachalcones, 9a 9f 9h, and 9i showed superior anti-inflammatory activity. Therefore,
protein expression of TLR4 and COX-2 was examined in RAW 264.7 cells pre-treated with 9a 9f 9h
–
i
–
i
,
,
,
,
,
or 9i before induction of inflammation. The results in Figure 9 show pre-treatment with these four
halo-substituted azachalcones attenuated the inflammatory responses induced by LPS. Compared
with the natural chalcones
1 and 2, these four synthetic compounds showed more significant inhibition
on inflammation than compound 2, but not compound 1.

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LPS
-LPS
LPS
a
9a
9f
9h
9i
1
2
TLR-4
β-actin
0.7
(a)
0.6
0.5
0.4
b
ab
ab
ab
0.3
0.2
0.1
ab
c
c
0
chalcone (10 μg/mL)
LPS (1 μg/mL)
-LPS
-
LPS
+
9a
+
9f
+
9h
+
9i
+
1
+
2
+
LPS
-LPS
LPS
9a
9f
9h
9i
1
2
COX-2
β-actin
1.6
1.4
(b)
a
b
ab
1.2
1.0
0.8
0.6
0.4
0.2
ab
ab
ab
c
c
0
chalcone (10 μg/mL)
-LPS
-
LPS
+
9a
+
9f
+
9h
+
9i
+
1
+
2
+
LPS (1 μg/mL)
Figure 9. Protein expression of (
synthetic halo-substituted azachalcones 9a
for 1 h before induction of inflammation by LPS (1
with the same letters are not significantly different (α = 0.05). LPS: lipopolysaccharide.
a
) TLR4 and (
b) COX-2. RAW 264.7 cells were pre-treated with
,
9f 9h
,
,
9i or natural synthetic compounds
1
or
2
(10
µg/mL)
µg/mL) for 6 h. Values indicate means
±
SD. Bars
It has been reported that the electron-withdrawing groups in B-ring, as well as the
electron-donating groups in A-ring on chalcone derivatives, enhanced the cytotoxic activity [30].
Since the C-2 OH group of chalcones involved intramolecular hydrogen bonding with the carbonyl

Molecules 2018, 23, 597
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group, however, the C-2 nitrogen atom of azachalcones 9a
–
i, were to lack this attractive force. Therefore,
we suspect that this difference may play a crucial role in their activity.
Based on the slight difference in structures between chalcones and azachalcones and comments
on cytotoxic activity of chalcones, we intended to realize the role of hydroxyl and nitrogen atom in
A ring of respective molecules. Therefore, we synthesized a series of monohalo substitutes in B ring
of both chalcones (7a–i) and azachalcone (9a–i). This paper is the first to report the comparison of
anti-inflammatory activity between synthetic halo-chalcones and -azachalcones. The results will lay the
background for future development of anti-inflammatory nutraceuticals. Evaluation of bioavailability
and toxicity of 9a, 9f, 9h, and 9i halo-azachalcones is taking place in our laboratory to pioneer in vivo
functional study.
3. Materials and Methods
Chemicals used for organic synthesis were purchased from commercial sources and used without
1
purification except when otherwise stated. H NMR (600 MHz) and 13C NMR (150 MHz) spectra were
recorded on a Bruker 600 instrument. Chemical shifts were reported in ppm and referenced to the
residue of solvent: (CDCl3: 7.26 ppm for 1H; 77.0 ppm for 13C). Melting points were determined on
Fargo MP-2D and not corrected. HRMS were recorded on Finnigan MAT-95S. Dulbecco’s modified
Eagle’s medium (DMEM), lipopolysaccharide (LPS), and dimethyl sulfoxide (DMSO) were purchased
from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and penicillin-streptomycin were
obtained from Biowest (Kancas, MO, USA). Anti-TLR4 (rabbit anti-toll-like receptor 4 polyclonal
antibody), anti-COX-2 (rabbit anti-cyclooxygenase-2 polyclonal antibody), and anti-β-actin (mouse
anti-beta actin monoclonal antibody) antibodies were purchased from Novus (Littleton, CO, USA).
Peroxidase-conjugated sheep anti-mouse IgG and goat anti-rabbit IgG were obtained from Jackson
ImmunoResearch (West Grove, PA, USA).
3.1. General Procedure of Claisen-Schmidt Condensation in Synthesis of 7a–i
To a stirred solution of 5 (0.100 mL, 0.108 g, and 0.890 mmol), for example, EtOH (2.5 mL) was
added the three equivalents of 8M KOH. This mixture was stirred at 0 ◦C for 10 min, followed by
addition of the corresponding aldehyde (1.5 equiv.) at that temperature. The mixture was gradually
warmed up to room temperature until
5 was consumed by TLC detection. At the end of reaction time,
the mixture was neutralized with 2N HCl, diluted with H2O, and extracted with CH2Cl2. The organic
layer was dried over MgSO4 and purified by flash column chromatography.
3.1.1. (E)-3-(4-Fluorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7a)
◦
Purification by flash column chromatography (CH2Cl2:Hexanes = 1:5–1:3). m.p. 118–120 C
1
(lit. [29] 120–122 ◦C). Yellow solid. H NMR (600 MHz, CDCl3)
δ
12.79 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H),
7.88 (d, J = 15.5 Hz, 1H), 7.66 (d, J = 5.6 Hz, 1H), 7.65 (d, J = 5.6 Hz, 1H), 7.58 (d, J = 15.5 Hz, 1H),
7.50 (t, J = 8.2 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 6.94
(t, J = 7.4 Hz, 1H). 13C NMR (150 MHz, CDCl3)
δ
193.5, 164.2 (1J = 251 Hz), 163.6, 140.1, 136.4, 130.8
2).
(
4J = 1.5 Hz), 130.6 (3J = 7.5 Hz) (
×
2), 129.6, 119.9, 119.8, 118.9, 118.6, 116.3, 116.2 (2J = 22.5 Hz) (
×
HRMS (ESI) calculated for C15H12FO2 [M + H]+ 243.0821. Found: 243.0823.
3.1.2. (E)-3-(4-Chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7b)
◦
Purification by flash column chromatography (CH2Cl2:Hexanes = 1:5–1:3). m.p. 152–154 C
(lit. [29] 143–145 C). Yellow solid. H NMR (600 MHz, CDCl3)
◦
1
δ 12.75 (s, 1H), 8.00 (d, J = 15.7 Hz, 1H),
7.92 (dd, J = 8.2, 1.7 Hz, 1H), 7.79 (d, J = 15.7 Hz, 1H), 7.66 (td, J = 7.6, 1.7 Hz, 1H), 7.51 (ddd, J = 8.5,
7.2, 1.6 Hz, 1H), 7.44–7.39 (m, 1H), 7.22 (td, J = 7.6, 1.0 Hz, 1H), 7.16 (ddd, J = 9.2, 8.2, 0.9 Hz, 1H), 7.04
(dd, J = 8.5, 1.0 Hz, 1H), 6.96 (td, J = 7.5, 1.1 Hz, 1H). 13C NMR (150 MHz, CDCl3)
δ 193.8, 163.7, 162.8,
161.1, 138.3, 136.5, 132.2, 130.2, 129.8, 124.6, 122.9, 120.0, 118.9, 118.7, 116.4. HRMS (ESI) calculated for
C15H12ClO2 [M]+ 259.0526. Found: 259.0530.

Molecules 2018, 23, 597
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3.1.3. (E)-3-(4-Bromophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7c)
◦
Purification ◦by flash column chromatography (CH2Cl2:Hexanes = 1:3–1:2). m.p. 145–148 C.
1
(lit. [29] 150–152 C). Yellow solid. H NMR (600 MHz, CDCl3)
δ 12.73 (s, 1H), 7.91 (d, J = 8.0 Hz,
1H), 7.85 (d, J = 15.5 Hz, 1H), 7.65 (d, J = 15.5 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.55–7.49 (m, 3H), 7.04
(d, J = 8.3 Hz, 1H), 6.95 (t, J = 7.3 Hz, 1H). 13C NMR (150 MHz, CDCl3) 193.5, 163.6, 144.0, 136.6, 133.5,
132.3, 130.0 (x3), 129.6, 125.3, 120.7, 119.9, 118.9, 118.7. HRMS (ESI) calculated for C15H12BrO2 [M + H]+
303.0021. Found: 303.0025.
3.1.4. (E)-3-(3-Fluorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7d)
Purification ◦by flash column chromatography (CH2Cl2:Hexanes = 1:8–1:6). m.p. 100–101 ◦C
1
(lit. [29] 109–110 C). Yellow solid. H NMR (600 MHz, CDCl3)
δ
12.70 (s, 1H), 7.91 (d, J = 8.1 Hz,
1H), 7.86 (d, J = 15.4 Hz, 1H), 7.64 (d, J = 15.4 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.44–7.40 (m, 2H), 7.36
(d, J = 9.6 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.04 (t, J = 8.3 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H). 13C NMR
(150 MHz, CDCl3) δ
193.4, 163.6, 163.0 (1J = 246 Hz), 143.9, 136.9, 136.7 (2J = 33 Hz), 130.6 (3J = 9 Hz),
129.6, 124.7, 121.4, 119.9 (
3), 118.8 (2J = 36 Hz), 117.7 (2J = 21 Hz), 114.6 (2J = 21 Hz). HRMS (ESI)
×
calculated for C15H12FO2 [M + H]+ 243.0821. Found: 243.0825.
3.1.5. (E)-3-(3-Chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7e)
◦
Purification by flash column chromatography (CH2Cl2:Hexanes = 1:5–1:3). m.p. 106–108 C
◦
(lit. [29] 105–107 C). Yellow-orange solid.1H NMR (600 MHz, CDCl3)
δ
12.71 (s, 1H), 7.91 (d, J = 8.0 Hz
,
1H), 7.83 (d, J = 15.5 Hz, 1H), 7.65 (s, 1H), 7.63 (d, J = 15.1 Hz, 1H), 7.52–7.49 (m, 2H), 7.40 (t, J = 7.9 Hz
,
1H), 7.37 (t, J = 7.9 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H). 13C NMR (150 MHz, CDCl3)
δ
193.4, 163.6, 143.6, 136.6, 136.4, 135.1, 130.7, 130.3, 129.7, 128.0, 127.0, 121.5, 119.9, 118.9, 118.7. HRMS
(ESI) calculated for C15H12ClO2 [M]+ 259.0526. Found: 259.0523.
3.1.6. (E)-3-(3-Bromophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7f)
◦
Purification by flash column chromatography (CH2Cl2:Hexanes = 1:5–1:3). m.p. 98–100 C
1
(lit. [29] 108–110 ◦C). Yellow solid. H NMR (600 MHz, CDCl3)
δ
12.70 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H),
7.83 (d, J = 15.5 Hz, 1H), 7.82 (s, 1H), 7.64 (d, J = 15.5 Hz, 1H), 7.56 (d, J = 7.6 Hz, 2H), 7.52 (t, J = 7.5 Hz,
1H), 7.32 (t, J = 7.9 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H). 13C NMR (150 MHz, CDCl3)
δ
193.4, 163.7, 143.6, 136.7 (x2), 133.6, 131.0, 130.5, 129.7, 127.5, 123.2, 121.5, 119.9, 119.0, 118.7. HRMS
(ESI) calculated for C15H12BrO2 [M + H]+ 303.0021. Found: 303.0023.
3.1.7. (E)-3-(2-Fluorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7g)
◦
Purification by flash column chromatography (CH2Cl2:Hexanes = 1:4–1:3). m.p. 90–93 C (lit. [46
]
80 C). Yellow solid. 1H NMR (600 MHz, CDCl3)
J = 15.7 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.51 (t, J = 7.3 Hz, 1H), 7.41 (dd, J = 13.6, 6.4 Hz, 1H), 7.22
(t, J = 7.6 Hz, 1H), 7.15 (dd, J = 10.6, 8.6 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.95 (t, J = 7.6 Hz, 1H). 13
δ 12.76 (s, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.78 (d,
◦
C
NMR (150 MHz, CDCl3)
δ
193.8, 163.6, 161.9 (1J = 254.0 Hz), 138.2, 136.5, 132.2 (3J = 8.8 Hz), 130.1,
129.7, 124.5 (4J = 3.0 Hz), 122.8 (3J = 7.9 Hz), 122.7, 120.0, 118.9, 118.6, 116.4 (2J = 23.0 Hz). HRMS (ESI)
calculated for C15H12FO2 [M + H]+ 242.0821. Found: 243.0825.
3.1.8. (E)-3-(2-Chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7h)
◦
Purification◦by flash column chromatography (CH2Cl2:Hexanes = 1:3–1:2). m.p. 103–105 C
1
(lit. [47] 138–140 C).Yellow solid. H NMR (600 MHz, CDCl3)
δ
12.72 (s, 1H), 8.31 (d, J = 15.6 Hz, 1H),
7.91 (d, J = 8.0 Hz, 1H), 7.77 (dd, J = 7.6, 1.3 Hz, 1H), 7.65 (d, J = 15.5 Hz, 1H), 7.52 (t, J = 7.3 Hz, 1H),
7.47 (d, J = 7.7 Hz, 1H), 7.36 (td, J = 8.1, 2.1 Hz, 1H), 7.34 (t, J = 6.9 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.95
(t, J = 7.9 Hz, 1H). 13C NMR (150 MHz, CDCl3)
δ 193.6, 163.7, 141.2, 136.5, 135.8, 133.0, 131.5, 130.4,

Molecules 2018, 23, 597
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129.7, 128.0, 127.1, 122.9, 119.9, 118.9, 118.7. HRMS (ESI) calculated for C15H12ClO2 [M + H]+ 259.0526.
Found: 259.0527.
3.1.9. (E)-3-(2-Bromophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (7i)
◦
Purification by flash column chromatography (CH2Cl2:Hexanes = 1:4–1:3). m.p. 103–105 C
1
(lit. [48] 103–104 ◦C). Yellow solid. H NMR (600 MHz, CDCl3)
δ 12.71 (s, 1H), 8.26 (d, J = 15.6 Hz,
1H), 7.90 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 15.4 Hz, 1H),
7.51 (td, J = 8.3, 1.1 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 7.27 (td, J = 7.6, 1.6 Hz, 1H), 7.04 (d, J = 8.3 Hz,
1H), 6.95 (t, J = 7.3 Hz, 1H). 13C NMR (150 MHz, CDCl3)
δ 193.6, 163.6, 143.7, 136.6, 134.8, 133.7, 131.6,
129.7, 127.8, 126.1, 123.0, 119.9, 118.9, 118.7. HRMS (ESI) calculated for C15H12BrO2 [M + H]+ 303.0021.
Found: 303.0019.
3.2. Biological Evaluation
3.2.1. Cell Culture
Mouse BALB/c macrophage cell line RAW 264.7, which was obtained from Biosource Collection
and Research Center of Food Industry Research and Development Institute (Shinchu, Taiwan),
was cultured in DMEM supplemented with FBS (1◦0%), sodium bicarbonate (0.22%), streptomycin
(100 units/mL), and penicillin (100 units/mL) in a 37 C incubator with 5% CO2. When 50% confluence
was reached, the cells were cultured with various synthetic chalcones for 1 h before LPS was added
to the medium (final concentration 1 µg/mL) to induce inflammation. Six hours later, the cells were
harvested for real-time PCR analysis and immunoblot analysis [49].
3.2.2. Cell Viability Assay
RAW 264.7 cells were seeded in 96-well plate at a concentration of 3
hours after seeding, the cells were treated with various concentrations of synthetic chalcones and LPS
(1 g/mL) for another 24 h before the medium was removed and the cells were cultured with new
×
105/well. Twenty-four
µ
medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at a final
concentration of 0.5 mg/mL. Four hours later, the purple formazan was dissolved by DMSO, and the
absorbance at 570 nm was taken. The absorbance is proportional to the viability of cells [49].
3.2.3. Quantitative Real-Time PCR Analysis
Effects of synthetic chalcones on the expressions of following genes were quantified: signal
recognition particle 72 (SRP 72), I
(IL-1 ), tumor necrosis factor- (TNF-
was extracted from the cells by using Aurum
Complementary DNA (cDNA) was synthesized by using iScript
Hercules, CA, USA) and performed on T3 Thermocycler (Biometra, Germany) with the program of
25 ◦C for 5 min, 42 ◦C for 30 min, and 85 ◦C for 5 min. The cDNA (5 ng/
L) was mixed with SYBR
green (iQ SYBR Green Supermix, BIO-RAD, Hercules, CA, USA) and primers (Table 3). The mixture
was subject to the thermal cycling under the following conditions: heating up to 95 C in 3 min,
κ
B-α
, cycloxygenase-2 (COX-2), interleukin-6 (IL-6), interleukin-1
), and inducible nitric oxide synthase (iNOS). Total RNA
Total RNA MiniKit (BIO-RAD, Hercules, CA, USA).
cDNA Synthesis Kit (BIO-RAD,
β
β
α
α
™
™
µ
™
◦
◦
◦
followed by 40 cycles at 95 C for 10 s and 50 C for 30 s. The expression of each gene was normalized
to the corresponding SRP 72 (internal reference) threshold cycle (ct) values by the 2−∆∆ct method [50].

Molecules 2018, 23, 597
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Table 3. Forward and reverse primers.
0
0
0 0
Reverse Primer 5 3
Gene
Forward Primer 5 3
SRP72
TLR4
IκB-α
p65
IL-1β
IL-6
CACACCCTAGCCCAACTTATT
TTCAGAACTTCAGTGGCTGGATTTA
CCTTCCTCAACTTCCAGAACAA
TGTGGAGATCATCGAACAGCCGAA
GTTACATCAGCACCTCACAA
CTTCCATCCAGTTGCCTTCT
TCAAGCGCCTCAACATCTAC
GTCTCCACAGCCACCAGATTCTC
GATCACAGCCAGCTTTCAGA
TGTTCCTGGTCCTGTGTAGCCATT
TTAGAAACAGCTCAGCCCATAC
CTCCGACTTGTGAAGTGGTATAG
GAGGTTGACTTTCTCCTGGTATG
CTTGAAGTGGGTCAGGATGTAG
TNF-α
COX-2
TTGTCTACTCCCAGGTTCTCT
CGGACTGGATTCTATGGTGAAA
3.2.4. Immunoblot Analysis
Cells were harvested and resuspended in lysis buffer containing 50 mM Tris-HCl, 1% Noniswr P40,
and 150 mM sodium chloride before centrifuged at 10,000 g for 30 min. Proteins in supernatant were
×
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to polyvinylidene fluoride (PVDF) membrane (PerkinElmer, Waltham, MA, USA). Five percent stacking
gel and 8% separating gel were used for SDS-PAGE. The membranes were blocked with 5% nonfat
dry milk in Tris-buffered saline containing Tween (TBST) (20 mM Tris-HCl, 137 mM NaCl, 0.1%
Tween-20, pH 8.3) for one hour before being incubated with primary antibody (anti-TLR4, anti-COX-2,
or anti-β
-actin) overnight at 4 ◦C and horseradish peroxidase-conjugated secondary antibody for 1 h
at room temperature. An enhanced chemiluminescence kit (ECL, PerkinElmer, Waltham, MA, USA)
was applied to detect the immunoreactive proteins [49].
4. Conclusions
A series of monohalo-substituted chalcones and azachalcones were synthesized and compared
their anti-inflammatory activity with a concentration of 10
more significant activity than chalcones on inhibition of pro-inflammatory factor expression, including
TLR4, I B- , p65, IL-1 , IL-6, TNF- , and COX-2. In lack of the internal hydrogen bonding between
C-2 hydroxy group and carbonyl group as in chalcones, our results indicate that the nitrogen atom in
µg/mL. In this series, azachalcones exerted
κ
α
β
α
azachalcones might be important to contribute their activity. Therefore, azachalcones 9a, 9f, 9h, and 9i
used for further investigation is undergoing in our laboratory.
Supplementary Materials: The following are available online. The 1H and 13C NMR data for compounds 7a
and 9a–i.
–i
Acknowledgments: We gratefully acknowledge the Ministry of Science and Technology (MOST 104-2113-M-032-002),
Tamkang University, and Shih Chien University for supporting this work.
Author Contributions: Tzenge-Lien Shih conceived and designed the experiments; Chia-Wai Li performed the
synthesis of compounds 7a–i and 9a–i; Ming-Hwa Liu performed the biological experiments. Chia-Feng Kuo
drsigned the biological experiments, analyzed the data, and wrote the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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