Concise synthesis and biological evaluation of chalcone derivatives bearing n-heterocyclic moieties

Article abstract of DOI:10.3987/COM-16-13452

In this study, we designed and synthesized a series of chalcone derivatives bearing N-heterocyclic moieties, and screened in vitro antiinflammatory in lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages and anticancer activity against a panel of hum

Full text of DOI:10.3987/COM-16-13452

HETEROCYCLES, Vol. 92, No. 6, 2016, pp. -. © 2016 The Japan Institute of Heterocyclic Chemistry
Received, 4th March, 2016, Accepted, 25th March, 2016, Published online, 5th April, 2016
DOI: 10.3987/COM-16-13452
CONCISE SYNTHESIS AND BIOLOGICAL EVALUATION OF
CHALCONE DERIVATIVES BEARING N-HETEROCYCLIC MOIETIES
Zewei Mao,a Xi Zheng,b Yuping Lin,a Yan Qi,b Chunyan Hu,a Chunping Wan,*b
and Gaoxiong Rao*a
a
School of Traditional Chinese Medicine, Yunnan University of Traditional
Chinese Medicine, Kunming, 650500, China; E-mail: rao13987124569@qq.com.
b
Central laboratory, The NO.1 Affiliated Hospital of Yunnan University of
Traditional Chinese Medicine, Kunming, 650021, China; E-mail:
wanchunping1012@163.com
Abstract – In this study, we designed and synthesized a series of chalcone
derivatives bearing N-heterocyclic moieties, and screened in vitro anti-
inflammatory in lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages
and anticancer activity against a panel of human tumor cell lines. The results
indicated that compound 9 not only had inhibitory effect on the generation of NO
(IC50=8.11 μM) and significantly inhibited the production of TNF-α, but also
showed better anticancer activity against Hela and SGC7901 (IC50=1.05 μM and
6.47 μM, respectively), which was identified as the most potent anti-inflammatory
and anticancer agent.
The secretion of inflammatory mediators by macrophages is involved in both the innate and adaptive
immune responses of many human diseases, such as infection and rheumatoid arthritis (RA).1 LPS, a
component of the cell wall of gram-negative bacteria, is known to activate a number of cellular signals in
macrophages. The macrophage produce many chemokines (NO, PGE2) and pro-inflammatory cytokines
(TNF-α, IL-6, IL-1β) when the macrophages are stimulated by LPS. So, the pro-inflammatory mediator is
considered as potential target in inflammatory-associated diseases. On the other hand, cancer is the
second most common cause of death in the world, which still remains a potentially life threatening
disease resulting from uncontrolled cell growth.
Chalcones play an important role in living organisms2 with a wide range of biological activities, which
are widely used as anti-inflammatory, antibacterial, antitumor and so on.3-6 Moreover, chalcones are
widely present in nature, and a number of anti-inflammatory and anticancer chalcones have been reported

in recent years, including sappanchalcone,7 butein,8 licochalcone A9 and echinatin.10 Due to their flexible
character bearing α, β-unsaturated ketone unit, chalcones are playing the vital role in drug research
through structural modification.11-14
Nitrogen heterocycles are important class of compounds having versatile biological activities, which are
used in drug design and synthesis generally as active units. The combination of the chalcone and
imidazole in one frame, has been reported in previous literature and is likely to lead to hybrid compounds
with potential activity.15 Moreover, in previous work, we have reported the synthesis of
chalcone-piperazine hybrid compounds and their potential anticancer activities.16 On the basis of these
results, we wanted to design and synthesize molecules towards the recombination of chalcone and active
nitrogen heterocycle moieties in the same structure through medicinal chemistry hybridization. Here, we
reported the concise synthesis of chalcone derivatives containing nitrogen heterocycle compound, and
screened the in vitro anti-inflammatory potential of compounds in LPS-stimulated inflammatory response
in RAW-264.7 macrophages and anticancer activity against a panel of human tumor cell lines by MTT
assay.
The general concise synthetic route used to obtain title compounds is outlined in Scheme 1. Treatment of
commercially available 4-dimethylaminobenzaldehyde with 4-fluoroacetophenone gave the 4-
dimethylamino-4’-fluorochalcone (1)17 in the presence of KOH resulted by the Aldol condensation, and it
was in trans-configuration. The desired compounds were formed from chalcone intermediate by
subsequent substitution reaction with different active nitrogen heterocycle compounds in the presence of
Cs2CO3 at 110 °C in DMF in good yields (2-16) following the literature procedure, and no aza-Michael
addition product was observed.18,19
Initially, K2CO3 was used in synthesis of title compounds. The results showed that compounds 8-16 were
obtained in satisfied yields, but compounds 2-7 were in low yields. Replaced by Cs2CO3, chalcone
derivatives 2-16 were in excellent yields，since the basicity of imidazole and benzimidazole was less than
that of other nitrogen active compounds.
Scheme 1. Concise synthesis of chalcone derivatives

Table 1. Structures and yields of compounds
Compd.
Het
M p (°C)
164-166
Yields (%) a Compd.
Het
M p (°C) Yields (%) a
N
N
91
71
162-164
85
2
10
11
N
N
N
N
165-167
166-168
179-181
179-181
86
3
4
5
N
N
79
76
161-163
85
12
N
N
164-166
174-176
77
65
13
14
N
180-181
180-182
67
69
6
7
HO
N
157-158
157-159
84
83
15
16
N
N
N
170-172
164-165
88
78
8
9
N
HN
N
a Yields represent isolated yields.
Comparative data for these compounds with respective to structures and yield are provided in Table 1. All
1
13
of the synthesized compounds were characterized by H NMR and C NMR, and some representative
compounds were characterized by HRMS analysis.
Anti-inflammatory activity: RAW 264.7 cell, a murine macrophage cell line, is widely used to establish
inflammatory model in vitro. In this study, we investigated the anti-inflammatory activity of synthetic
derivatives in LPS-induced RAW 264.7 on the generation of NO and pro-inflammatory cytokines.
To evaluate the effect of chalcone analogs (2-16) on NO generation, the RAW 264.7 cells were
stimulated with LPS for 24 h, the level of NO from cell culture supernatant was measured by Griess
reagent. The results indicated that compounds 8 and 9 showed significant inhibitory effect on the
generation of NO in LPS-induced RAW 264.7, the IC50 value was calculated to be about 10.13 μM and
8.11 μM respectively (Table 2), neither had showed obvious cell cytotoxicity to RAW 264.7 cells at the
concentration of 100 μM.
Stimulated macrophages have great impact on inflammatory diseases via excess production of cytokines,
including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and other inflammatory
mediators such as nitric oxide (NO). In order to detect the effect of compound on pro-inflammatory
cytokines (TNF-α, IL-6), we then selected the compound 9 for further study. The production of TNF-α in
LPS-induced RAW 264.7 was significantly down-regulated after exposed to indicate concentration of
compound 9. However, interestingly, it had no apparent effects on the generation of IL-6 (Figure. 1). At

present, our results indicated that compound 9 significantly inhibited the production of TNF-α and NO in
LPS-induced RAW264.7.
Table 2. Anti-inflammatory activities of compounds
Compd.
NO generation (IC50, μM) a Compd.
NO generation (IC50, μM) a
17.12
21.35
30.72
>40
16.68
26.10
>40
2
3
4
5
6
7
10
11
12
13
14
15
16
>40
>40
19.07
>40
>40
10.13
8.11
>40
8
9
a Values represent the concentration required to produce 50% inhibition of the response.
Figure. 1 The effect of compound 9 on TNF- and IL-6 production in LPS -induced RAW 264.7. The RAW 264.7 cell was stimulated with
LPS in the present or absence of various concentration of compound 9 for 24h, the culture supernatant was harvested to measure the
production of TNF- and IL-6 by ELISA. The results were expressed as mean ± s.e.m with three independent experiments. * P < 0.05, ** P
< 0.01, ***P < 0.001 versus control group.
Anticancer activity: Cytotoxic activity of novel synthesized hybrid derivatives were evaluated against
human lung cancer cell line A549, human cervical carcinoma Hela and human gastric carcinoma
SGC7901 by MTT assay, cisplatin (DDP) was used as reference drug. The biological results of hybrid
compounds are summarized in Tables 3.
As shown in Table 3, the N-heterocycle moieties of the hybrid compounds have an obvious influence on
the antitumor activities. To our delight, compounds bearing imidazole, triazole, piperazine or
3-hydroxyazetidine displayed potent or similar cytotoxic activity against human tumor cell lines
compared to DDP. Compound 2 displayed potent anticancer activity against A549 (10.21 μM) and
Hela (7.42 μM), compound 8 appeared good cytotoxic activity against SGC7901 (9.53 μM), and
compound 11 showed potent anticancer activity against Hela (6.58 μM). Especially, among all
derivatives, compound 9 showed the best IC50 value against Hela (1.05 μM) and SGC7901 (6.47 μM),
which was identified as the most potent anticancer agent. However, substitution at the N-heterocycle with

other substituted groups resulted in lower activity. For example, compounds 3-4 and 10-11 were less
active than compounds 2 and 9, respectively.
Table 3. In vitro cytotoxic activities of compounds
Cell lines (IC50, μM) a
Cell lines (IC50, μM) a
Compd.
Compd.
A549
10.21
21.64
>40
Hela
7.42
17.11
>40
SGC7901
A549
>40
Hela
12.53
6.58
SGC7901
>40
19.34
25.26
>40
2
10
>40
17.05
28.78
>40
3
4
5
6
7
8
9
11
>40
>40
12
26.50
33.14
>40
>40
19.73
24.82
>40
25.16
13.01
>40
>40
13
>40
7.72
>40
14
>40
>40
27.24
>40
15
>40
16.23
1.05
9.53
6.47
23.21
11.54
>40
16
>40
20.52
12.44
DDP
a Each value was reproduced in triplicate.
Finally, we discussed a clear tendency of structure-activity relationship: Comparison of the activities of
the chalcone derivatives 8 and 9 revealed that compounds bearing piperazine or triazole moiety showed
significant anti-inflammatory activity, and compounds 2, 8, 9, 11 and 14 bearing imidazole, triazole,
piperazine or 3-hydroxyazetidine moiety displayed potent antitumor activity. Interestingly, both
anti-inflammatory and anticancer activity would be decreased when substitution at the N-heterocycle
moiety with other groups except pyrimidyl. Further derivatization and biological research is under
progress in our laboratory and will be reported in due course.
EXPERIMENTAL
Starting materials were commercially available and analytically pure. Melting points were measured on
1
YANACO microscopic melting point meter (Yanaco, Japan) and were uncorrected. H NMR and 13C
NMR spectras were recorded on a Bruker AV 300 spectrometer (Bruker Company, Germany), using
TMS as internal standard and CDCl3 as solvent, respectively. TLC analysis was carried out on silica gel
plates GF254 (Qindao Haiyang Chemical, China). High-resolution mass spectra were performed on an
ESI Q-TOF MS spectrometer (Micromass, England).
Synthesis of 4-Dimethylamino-4’-fluorochalcone (1): To a solution of EtOH (15 mL), 4-dimethyl-
aminobenzaldehyde (1.49 g, 10 mmol) and 4-fluoroacetophenone (1.38 g, 10 mmol), was added 20%
KOH (10mL) and left to react for 16 h at rt. The reaction was quenched by the addition of water (30 mL)
and was extracted with CHCl3 (3×20 mL). The organic layer was dried using anhydrous sodium sulfate,
concentrated in vacuo and purified by column chromatography (10% EtOAc/PE, 2.35g, 87% yield).
Yellow solid; mp 139-141C; 1HNMR (300 MHz, CDCl3) δ: 8.01-8.05 (m, 2H), 7.81 (d, J=15.3 Hz, 1H),
7.55 (d, J=8.7 Hz, 2H), 7.32 (d, J=15.2 Hz, 1H), 7.11-7.17 (m, 2H), 6.69 (d, J=8.7Hz, 2H), 3.03 (s, 6H);

13C NMR (75 MHz，CDCl3) δ: 188.9, 166.9, 163.6, 152.1, 146.0, 135.4, 130.9, 130.7, 130.5, 122.5, 116.4,
115.6, 115.3, 111.8, 40.1.
General procedure for the preparation of chalcone derivatives 2-16. To a stirred solution of the
4-dimethylamino-4’-fluorochalcone 1 (1.0 mmol) and Cs2CO3 (2.0 mmol) in dried DMF (5 mL), nitrogen
heterocycle (1.5 mmol) was added and reaction mixture was stirred for 12-15 h at 110 °C. After
completion of the reaction as indicated by TLC, The reaction was quenched by the addition of CHCl3 (20
mL) and was washed with water (3×20 mL). The organic layer was dried using anhydrous sodium sulfate,
concentrated in vacuo and purified by column chromatography to afford 2-16 in 55-91% yields.
4-Dimethylamino-4’-(1-imidazolyl)chalcone (2). Yellow solid; 1H NMR (300 MHz, CDCl3) δ: 8.14 (d,
J=8.7Hz, 2H), 7.95 (s, 1H), 7.85 (d, J=15.3 Hz, 1H), 7.57 (d, J=8.7 Hz, 2H), 7.51 (d, J=8.4 Hz, 2H), 7.36
(d, J=4.5 Hz, 2H), 7.24 (s, 1H), 6.71 (d, J=8.7 Hz, 2H), 3.05 (s, 6H); 13C NMR (75 MHz, CDCl3) δ: 188.8,
152.3, 146.5, 140.0, 137.9, 135.4, 131.0, 130.6, 130.2, 122.4, 120.7, 117.8, 116.1, 111.8, 40.1; HRMS:
m/z calcd for C20H20N3O (M+H)+ 318.1601, found 318.1601.
1
4-Dimethylamino-4’-(2-methyl-1-imidazolyl)chalcone (3). Yellow solid; H NMR (300 MHz, CDCl3)
δ: 8.11 (d, J=5.7 Hz, 2H), 7.90 (d, J=8.4 Hz, 1H), 7.55 (d, J=8.4 Hz, 2H), 7.34-7.47 (m, 2H), 7.04 (s, 2H),
6.62-6.71 (m, 3H), 3.01 (s, 6H), 2.40 (s, 3H); 13C NMR (75 MHz, CDCl3) δ: 186.8, 158.5, 153.8, 152.2,
144.8, 141.3, 138.3, 131.2, 130.9, 129.5, 128.4, 125.4, 125.3, 125.0, 122.5, 120.6, 113.7, 112.1, 111.2,
40.3, 14.3.
1
4-Dimethylamino-4’-(2-ethyl-1-imidazolyl)chalcone (4). Brown solid; H NMR (300 MHz, CDCl3) δ:
8.13 (d, J=5.1 Hz, 2H), 7.87 (d, J=15.6 Hz, 1H), 7.58 (d, J=9.0 Hz, 2H), 7.28-7.42 (m, 3H), 7.10 (d,
J=15.6 Hz, 2H), 6.71 (d, J=9.0 Hz, 2H), 3.05 (s, 6H), 2.76 (q, J=7.5 Hz, 2H), 1.30 (t, J=7.5 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ: 189.2, 152.3, 149.5, 146.7, 140.8, 138.7, 130.7, 129.6, 128.0, 125.5, 122.3,
120.4, 116.1, 111.8, 40.1, 20.7, 12.4.
4-Dimethylamino-4’-(1-benzimidazolyl)chalcone (5). Pale yellow solid; 1H NMR (300 MHz, CDCl3) δ:
8.19 (d, J=8.4 Hz, 2H), 8.15 (s, 1H), 7.86-7.90 (m, 1H), 7.81 (s, 1H), 7.51 - 7.60 (m, 5H), 7.31-7.36 (m,
3H), 6.66 (d, J=9.0 Hz, 2H), 2.99 (s, 6H); 13C NMR (75 MHz, CDCl3) δ: 188.7, 152.2, 146.5, 144.3,
142.0, 139.3, 138.2, 133.2, 130.6, 130.2, 124.0, 123.2, 123.1, 122.3, 120.7, 115.9, 111.8, 110.6, 40.0.
1
4-Dimethylamino-4’-(2-methyl-1-benzimidazolyl)chalcone (6). Yellow solid; H NMR (300 MHz,
CDCl3) δ: 8.24 (d, J=8.4 Hz, 2H), 7.91 (d, J=15.6 Hz, 1H), 7.78 (d, J=7.8 Hz, 1H), 7.61 (d, J=9.0 Hz, 2H),
7.53 (d, J=8.7 Hz, 2H), 7.49 (d, J=15.6 Hz, 1H), 7.20-7.30 (m, 3H), 6.73 (d, J=8.7 Hz, 2H), 3.07 (s, 6H),
2.57 (s, 3H); 13C NMR (75 MHz, CDCl3) δ: 189.3, 152.3, 146.8, 142.7, 139.3, 130.7, 130.0, 126.9, 122.9,
122.7, 119.2, 116.1, 111.8, 109.9, 40.1, 14.6.
4-Dimethylamino-4’-(5, 6-dimethyl-1-benzimidazolyl)chalcone (7). Brown solid; 1H NMR (300 MHz,
CDCl3) δ: 8.22 (d, J=8.4 Hz, 2H), 8.07 (s, 1H), 7.88 (d, J=15.6 Hz, 1H), 7.55-7.64 (m, 5H), 7.39 (s, 2H),

6.71 (d, J=8.7 Hz, 2H), 3.04 (s, 6H), 2.40 (s, 6H); 13C NMR (75 MHz, CDCl3) δ: 189.0, 152.2, 146.6,
142.9, 141.2, 139.7, 138.0, 133.3, 132.1, 131.7, 130.7, 130.2, 123.1, 122.4, 120.7, 116.0, 111.8, 110.7,
40.1, 20.7, 20.3.
4-Dimethylamino-4’-(1-triazolyl)chalcone (8). yellow solid; 1H NMR (300MHz, CDCl3) δ: 8.67 (s, 1H),
8.17 (s, 1H), 8.14 (s, 2H), 7.85 (d, J=15.6 Hz, 1H), 7.84 (d, J=8.7 Hz, 2H), 7.57 (d, J=8.7 Hz, 2H), 7.35
13
(d, J=15.6 Hz, 1H), 6.71 (d, J=8.7 Hz, 2H), 3.05 (s, 6H); C NMR (75 MHz, CDCl3) : 188.8, 152.9,
152.3, 146.6, 141.0, 139.4, 138.5, 130.6, 130.1, 122.4, 119.4, 116.1, 111.8, 40.1; HRMS: m/z calcd for
C19H19N4O (M+H)+ 319.1553, found 319.1554.
1
4-Dimethylamino-4’-(1-piperazinyl)chalcone (9). brown solid; H NMR (300 MHz, CDCl3) δ: 8.01 (d,
J=9.0 Hz, 2H), 7.80 (d, J=15.3 Hz, 1H), 7.56 (d, J=9.0 Hz, 2H), 7.41 (d, J=15.6 Hz, 1H), 6.92 (d, J=9.0
Hz, 2H), 6.70 (d, J=9.0 Hz, 2H), 3.33 (t, J=4.8 Hz, 4H), 3.01-3.04 (m, 10H), 2.12 (s, 1H); 13C NMR (75
MHz, CDCl3) : 188.3, 154.2, 151.8, 144.1, 130.3, 130.1, 129.1, 123.0, 116.8, 113.6, 111.8, 48.5, 45.9,
40.2; HRMS: m/z calcd for C21H26N3O (M+H)+ 336.2070, found 336.2072.
4-Dimethylamino-4’-(N-methyl-1-piperazinyl)chalcone (10). Brown solid; 1H NMR (300 MHz, CDCl3 )
δ: 8.00 (d, J=9.0 Hz, 2H), 7.80 (d, J=15.6 Hz, 1H), 7.55 (d, J=8.7 Hz, 2H), 7.39 (d, J=15.6 Hz, 1H), 6.92
(d, J=8.7 Hz, 2H), 6.70 (d, J=9.0 Hz, 2H), 3.38 (t, J=5.1 Hz, 4H), 3.01 (s, 6H), 2.57 (t, J=5.1 Hz, 4H),
2.34 (s, 3H); 13C NMR (75 MHz, CDCl3) : 188.3, 153.8, 151.8, 144.1, 130.3, 130.1, 129.2, 123.1, 116.9,
113.7, 111.9, 54.8, 47.3, 46.1, 40.1.
4-Dimethylamino-4’-(N-(2-pyrimidyl)-1-Piperazinyl)chalcone (11). Yellow solid; 1H NMR (300 MHz,
CDCl3 ) δ: 8.35 (d, J=4.8 Hz, 2H), 8.03 (d, J=8.7 Hz, 2H), 7.81 (d, J=15.6 Hz, 1H), 7.56 (d, J=8.7 Hz,
2H), 7.40 (d, J=15.3 Hz, 1H), 6.96 (d, J=9.0 Hz, 2H), 6.70 (d, J=9.0 Hz, 2H), 6.55 (t, J=4.8 Hz, 1H), 4.01
(t, J=5.4 Hz, 4H), 3.47 (t, J=5.4 Hz, 4H), 3.02 (s, 6H); 13C NMR (75 MHz, CDCl3) : 188.4, 161.6, 157.8,
153.7, 151.8, 144.2, 130.4, 130.1, 129.4, 123.1, 116.9, 113.8, 111.9, 110.3, 47.3, 43.3, 40.1.
4-Dimethylamino-4’-(1-pyrazolyl)chalcone (12). Yellow solid; 1H NMR (300 MHz, CDCl3 ) δ: 8.14 (d,
J=8.7 Hz, 2H), 8.02 (d, J=2.1 Hz, 1H), 7.77-7.85 (m, 4H), 7.58 (d, J=8.7 Hz, 2H), 7.38 (d, J=15.3 Hz,
13
1H), 6.70 (d, J=8.7 Hz, 2H), 6.52 (t, J=1.8 Hz, 1H), 3.04 (s, 6H); C NMR (75 MHz, CDCl3) : 189.0,
152.1, 146.0, 142.7, 141.9, 136.7, 130.5, 129.9, 126.9, 122.5, 118.4, 116.3, 111.8, 108.4, 40.1; HRMS:
m/z calcd for C20H20N3O (M+H)+ 318.1601, found 318.1603.
1
4-Dimethylamino-4’-(3,5-dimethyl-1-pyrazolyl)chalcone (13). Yellow solid; H NMR (300 MHz,
CDCl3 ) δ: 8.11 (d, J=8.7 Hz, 2H), 7.84 (d, J=15.6 Hz, 1H), 7.59 (d, J=6.6 Hz, 2H),, 7.57 (d, J=7.2 Hz,
2H), 7.36 (d, J=15.6 Hz, 1H), 6.71 (d, J=8.7 Hz, 2H), 6.04 (s, 1H), 3.04 (s, 6H), 2.38 (s, 3H), 2.32 (s, 3H);
13C NMR (75 MHz, CDCl3) : 189.6, 152.1, 149.8, 146.3, 142.9, 139.6, 137.2, 130.5, 129.3, 123.8, 122.5,
116.6, 111.8, 108.1, 40.1, 13.6, 12.8.

1
4-Dimethylamino-4’-(3-hydroxyazetidin-1-yl)chalcone (14). Pale yellow solid; H NMR (300 MHz,
CDCl3 ) δ: 7.94 (d, J=8.7 Hz, 2H), 7.78 (d, J=15.3 Hz, 1H), 7.54 (d, J=8.7 Hz, 2H), 7.38 (d, J=15.3 Hz,
1H), 6.70 (d, J=8.7 Hz, 2H), 6.40 (d, J=8.7 Hz, 2H), 4.77-4.81 (m, 1H), 4.27 (t, J=8.1 Hz, 2H), 3.79-3.83
(dd, J=4.5 Hz, 4.5Hz, 2H), 3.03 (s, 6H); HRMS: m/z calcd for C20H23N2O2 (M+H)+ 323.1754, found
323.1750.
1
4-Dimethylamino-4’-(1-pyrrolyl)chalcone (15). Yellow solid; H NMR (300 MHz, CDCl3 ) δ: 8.00 (d,
J=9.0 Hz, 2H), 7.79 (d, J=15.3 Hz, 1H), 7.54 (d, J=8.7 Hz, 2H), 7.43 (d, J=15.3 Hz, 1H), 6.68 (d, J=9.0
13
Hz, 2H), 6.53 (d, J=8.7 Hz, 2H), 3.34 (t, J=6.9 Hz, 4H), 2.98 (s, 6H), 1.96-2.01 (m, 4H); C NMR (75
MHz, CDCl3) : 187.9, 151.6, 150.7, 143.2, 130.7, 130.0, 126.2, 123.4, 117.2, 111.9, 110.9, 47.6, 40.2,
25.4.
1
4-Dimethylamino-4’-(1-piperidyl)chalcone (16). Brown solid; H NMR (300 MHz, CDCl3 ) δ: 7.99 (d,
J=9.0 Hz, 2H), 7.80 (d, J=15.6 Hz, 1H), 7.55 (d, J=8.7 Hz, 2H), 7.41 (d, J=15.6 Hz, 1H), 6.90 (d, J=8.7
13
Hz, 2H), 6.69 (d, J=8.7 Hz, 2H), 3.35 (s, 4H), 3.01 (s, 6H), 1.65 (s, 6H); C NMR (75 MHz, CDCl3) :
188.2, 154.2, 143.8, 130.5, 130.0, 128.2, 123.3, 117.0, 113.5, 111.9, 48.7, 40.2, 25.4, 24.4.
Antimicrobial Activity. Briefly, murine RAW264.7 macrophages (American Type Culture Collection,
Manassas) were plated in 96 well plate at a density of 1× 105 cells/well and stimulated with 1μg/mL LPS
in the present or absence of various concentration of compound for 24 h, the culture supernatant were
harvested to measure NO and cytokines production. The production of NO was determined by assaying
culture supernatant for NO2-, a stable reaction product of NO, 100 μL of supernatant was mixed with an
equal volume of Griess reagent [1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine
dihydrochloride in 2.5% H3PO4] at room temperature for 10 min. Absorbance was measured at 540 nm in
a microplate reader. Nitrite concentration was calculated from a NaNO2 standard curve. For the cytokines
detection, culture supernatants were harvested at 36 h to measure IL-6 and TNF-α level by ELISA
following the manufacture’s instruction.
Antitumor activity: About 1×104 cell/well were seeded into 96-well microtiter plates. After 24 h
post-seeding, cells were treated with vehicle control or various concentrations of samples for 48 h. 20 μL
of MTT solution (5 mg/mL) was added to each well and the tumor cells were incubated at 37 °C in a
humidified atmosphere of 5% CO2 air for 4 h. Upon removal of MTT/medium, 150 μL of DMSO was
added to each well and the plate was agitated at oscillator for 5 min to dissolve the MTT-formazan. The
assay plate was read at a wavelength of 570 nm using a microplate reader.
ACKNOWLEDGEMENTS
This work was financially supported by the Natural Science Foundation of China (81460624) and the
Application Basic Research Program of Yunnan Province (2014FZ087).

REFERENCES
1. W. L. Smith and L. J. Marnett, Biochim. Biophys. Acta, 1991, 1083, 1.
2. F. Berrue, M. W. B. McCulloch, and R. G. Kerr, Bioorg. Med. Chem., 2011, 19, 6702.
3. L. Jayasinghe, B. A. I. S. Balasooriya, W. C. Padmini, N. Hara, and Y. Fujimoto, Phytochemistry,
2004, 65, 1287.
4. K. Sugamoto, Y. I. Matsusita, K. Matsui, C. Kurogi, and T. Matsui, Tetrahedron, 2011, 67, 5346.
5. N. Mishra, P. Arora, B. Kumar, L. C. Mishra, A. Bhattacharya, S. K. Awasthi, and V. K. Bhasin, Eur.
J. Med. Chem., 2008, 43, 1530.
6. Z. Nowakowska, B. Kedzia, and G. Schroeder, Eur. J. Med. Chem., 2008, 43, 707.
7. G. S. Jeong, D. S. Lee, B. Li, H. J. Lee, E. C. Kim, and Y. C. Kim, Eur. J. Pharmacol., 2010, 644, 1.
8. Z. Wang, Y. Y. Lee, J. S. Eun, and E. J. Bae, Eur. J. Pharmacol., 2014, 738, 40.
9. M. Chen, S. B. Christensen, J. Blom, E. Lemmich, L. Nadelmann, K. Fich, T. G. Theander, and A.
Kharazmi, Antimicrob. Agents Chemother., 1993, 37, 2550.
10. S. I. Ayabe and T. Furuya, Tetrahedron Lett., 1981, 22, 2097.
11. J. Chen, J. Yan, J. H. Hu, Y. Q. Pang, L. Huang, and X.S. Li, RSC Adv., 2015, 5, 68128.
12. Y. N. Shen, L. Lin, H.Y. Qiu, W. Y. Zou, Y. Qian, and H. L. Zhu, RSC Adv., 2015, 5, 23767.
13. K. V. Sashidhara, M. Kumar, R. K. Modukuri, R. Sonkar, G. Bhatia, A. K. Khanna, S. Rai, and R.
Shukla, Bioorg. Med. Chem. Lett., 2011, 21, 4480.
14. C. Tang, L. H. Zhu, Y. Chen, R. Qin, Z. N Mei, J. Xu, and G. Z. Yang, RSC Adv., 2014, 4, 10862.
15. V. K. Marrapu, V. Chaturvedi, S. Singh, S. Singh, S. Sinha, and K. Bhandari, Eur. J. Med. Chem.,
2011, 46, 4302.
16. Z. W. Mao, X. Zheng, Y. Qi, M. D. Zhang, Y. Huang, C. P. Wan, and G. X. Rao, RSC Adv., 2016, 6,
7723.
17. A. Kamal, A. Mallareddy, P. Suresh, V. L. Nayak, R. V. C. R. N. C. Shetti, N. S. Rao, J. R. Tamboli, T.
B. Shaik, M. V. P. S. Vishnuvardhan, and S. Ramakrishna, Eur. J. Med. Chem., 2012, 47, 530.
18. A. J. Watson and A. J. Fairbanks, Eur. J. Org. Chem., 2013, 30, 6784.
19. M. L. Kantam, J. Yadav, S. Laha, B. Sreedhar, and S. Jha, Adv. Synth. Catal., 2007, 349, 1938.