Synthesis and biological evaluation of chalcone derivatives containing aminoguanidine or acylhydrazone moieties

Article abstract of DOI:10.1016/j.bmcl.2016.11.001

Three novel series of chalcone derivatives containing an aminoguanidine or acylhydrazone moiety were designed, synthesized and evaluated in terms of their antibacterial, antifungal and anti-inflammatory activities. Most of the synthesized compounds showed

Full text of DOI:10.1016/j.bmcl.2016.11.001

Accepted Manuscript
Synthesis and biological evaluation of chalcone derivatives containing amino-
guanidine or acylhydrazone moieties
Zhi-Yu Wei, Ke-Qiang Chi, Zhan-Kui Yu, Hong-Yan Liu, Liang-Peng Sun,
Chang-Ji Zheng, Hu-Ri Piao
PII:
S0960-894X(16)31141-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.001
BMCL 24396
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 July 2016
28 October 2016
1 November 2016
Please cite this article as: Wei, Z-Y., Chi, K-Q., Yu, Z-K., Liu, H-Y., Sun, L-P., Zheng, C-J., Piao, H-R., Synthesis
and biological evaluation of chalcone derivatives containing aminoguanidine or acylhydrazone moieties, Bioorganic
& Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.001
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Synthesis and biological evaluation of chalcone derivatives
containing aminoguanidine or acylhydrazone moieties
Zhi-Yu Wei a, Ke-Qiang Chi b, Zhan-Kui Yu a, Hong-Yan Liu a, Liang-Peng Sun a,
Chang-Ji Zheng a,*, Hu-Ri Piao a,*
a Key Laboratory of Natural Resources and Functional Molecules of the Changbai
Mountain, Affiliated Ministry of Education; Yanbian University College of Pharmacy,
Yanji, Jilin Province, 133002, PR China
b Medical College of Dalian University, Dalian, Liaoning Province, 116622, PR China
*Correspondence Author: Tel: + 86(433)243-5003; Fax: + 86(433)243-5004
E-mail: piaohuri@yahoo.com.cn (H. R. Piao)
*Correspondence Author: Tel: + 86(433)243-6015; Fax: + 86(433)243-5026
E-mail: zhengcj@ybu.edu.cn (C. J. Zheng)
1

Abstract
Three novel series of chalcone derivatives containing an aminoguanidine or
acylhydrazone moiety were designed, synthesized and evaluated in terms of their
antibacterial, antifungal and anti-inflammatory activities. Most of the synthesized
compounds showed potent inhibitory activity towards various bacteria and one fungus
with minimum inhibitory concentrations (MICs) ranging from 1 to 8 µg/mL.
Compared with our previously reported chalcone derivatives (MICs > 64 µg/mL),
these compounds exhibited improved antibacterial activities (MICs = 2 µg/mL)
against Gram-negative bacterial strains (Escherichia coli 1924 and 1356). Compounds
4f and 4h were found to be the most potent with an MIC value of 1 µg/mL against the
Gram-negative bacterial strains Salmonella typhimurium 1926 and the fungus
Candida albicans 7535. In addition, compound 4f displayed the most potent
anti-inflammatory activity of all of the compounds prepared in the current study with
92.45% inhibition after intraperitoneal administration, making it more potent than the
reference drugs indomethacin and ibuprofen. The cytotoxic activity of the compound
4f was assessed in HeLa, Hep3B and L02 cells.
Keywords: Chalcone; Acylhydrazone; Aminoguanidine; Antibacterial activity;
Antifungal activity; Anti-inflammatory activity; Cytotoxicity
2

Numerous research groups throughout the world are currently involved in an
urgent search for novel antibacterial and antifungal agents to overcome the emergence
of new infectious diseases and the increasing number of multidrug-resistant microbial
pathogens.1,2 Although several classes of antimicrobial agents are currently available,
pathogenic bacteria and fungi have developed resistance to these drugs in the majority
of cases.3,4 Additionally, there is a strong relationship between bacterial infection and
inflammation.5 Furthermore, inflammation remains a common and poorly controlled
clinical problem that can be life threatening in extreme cases, including allergic
reaction, autoimmune disease and organ rejection following transplantation surgery.6
There is, therefore, an urgent need to develop novel antibacterial, antifungal and
anti-inflammatory agents to address these issues.
Chalcones belong to the flavonoid class of natural products and have attracted
considerable interest because of their relatively simple structures and wide variety of
pharmacological activities.7-11 For example, chalcone-based compounds have been
reported to exhibit anticancer,12 anti-inflammatory,13 anti-ulcerative,14 analgesic,15
antiviral,16 antifungal,17 antimalarial18 and antibacterial activities.19 Aminoguanidine
derivatives have recently captured the attention of numerous researchers because of
their diverse range of biological properties, including their antibacterial,20 antifungal21
and anti-inflammatory activities.22 Acylhydrazones have also received considerable
interest from researchers working in a variety of different fields because they possess
a broad range of pharmacological properties, including antibacterial23,24 and
anticancer activities.25 In our previous work, we reported the development of a series
of chalcone derivatives that showed potent activity against Gram-positive bacterial
strains, including multidrug-resistant clinical isolates.26 Unfortunately, none of these
compounds are active against Gram-negative bacteria. Aminoguanidine derivatives
were reported as antifungal agents with a potency of 100 µg/mL.21 In a continuation
of our research towards the discovery and development of increasingly potent
antibacterial agents, we report herein the structure-based design of chalcone
derivatives containing an aminoguanidine or acylhydrazone moiety. In this way, we
have developed three novel series of chalcone derivatives (Figure 1), totaling 26
3

compounds, which were designed, synthesized and screened for their antibacterial,
antifungal and anti-inflammatory activities in vitro. The substituents on the phenyl
ring were simultaneously changed to investigate their contribution to the biological
activity.
Figure 1. The structures of the target compounds.
N
H
N
N
O
5a-g
R
O
N
H
N
b
NH2
O
CHO
H
N
CHO
NH2
NH
N
OHC
2
b
a
NH
NH2
R
R
R
H2N
O
O
O
N
H
H2CO3
1
3
4a-l
H
N
b
NH2
R1
O
H
N
N
R1
O
R
O
6a-g
Scheme 1. Synthetic scheme for the synthesis of the target compounds. Reagents and conditions:
(a) NaOH, EtOH, 23°C, 3-4 h; (b) Aminoguanidine bicarbonate or isoniazide or benzoylhydrazine,
EtOH, HCl, 50-60 oC, reflux, 8-12 h.
The route used for the synthesis of compounds 4a–l, 5a–g and 6a–g is shown in
Scheme 1. The key intermediate 3 was prepared by the Claisen–Schmidt condensation
of terephthalaldehyde (2) with a substituted acetophenone (1) using a previously
4

described method.26,27 Compounds 4a–l were prepared by the reaction of 3 with
aminoguanidine bicarbonate in the presence of concentrated hydrochloric acid in
refluxing ethanol. Compounds 5a–g and 6a–g were prepared by the reactions of
compound 3 with isonicotinic acid hydrazide and substituted benzoyl hydrazine,
respectively, in the presence of catalytic amounts of hydrochloric acid in ethanol.22
The structures of the synthesized compounds were characterized by IR, 1H NMR, 13C
NMR, HRMS and MS analyses.28
The in vitro antimicrobial and antifungal activities of the synthesized compounds
were evaluated against a variety of strains of bacteria and one fungus (including
multidrug-resistant clinical isolates), using a 96-well microtiter plate screening format
to obtain their minimum inhibitory concentration (MIC) values.29 Gatifloxacin,
moxifloxacin, fluconazole, norfloxacin and oxacillin were used as positive controls.
The compounds were screened against four Gram-positive strains (S. aureus 4220, S.
aureus 209, S. aureus 503 and Streptococcus mutans 3065), four clinical isolates of
multidrug-resistant Gram-positive bacterial strains (MRSA 3167 and 3506, and
QRSA 3505 and 3519), four Gram-negative strains (Escherichia coli 1924, E. coli
1356, Salmonella typhimurium 1926 and Pseudomonas aeruginosa 2742) and one
fungus (Candida albicans 7535).
Table 1. Inhibitory activities (MICa, µg/mL) of compounds 4a–l, 5a–g and 6a–g against various
bacteria.
N
H
N
H
N
H
N
NH2
NH
R1
N
N
N
R
O
O
R
R
O
O
O
6a-g
4a-l
5a-g
Gram-positive strains
Gram-negative strains
Fungus
7535j
Compd
R
R1
4220b 503c 209d 3065e 1924f 1356g 1926h 2742i
4a
4b
4c
4d
4e
4f
2-F
4-F
—
—
—
—
—
—
—
—
4
4
8
4
2
2
4
2
8
8
8
4
4
2
8
2
8
8
8
4
4
2
8
2
8
8
8
4
4
2
8
2
8
4
8
4
2
2
8
2
16
8
4
4
8
2
4
1
4
1
4
4
8
4
2
2
8
2
8
4
8
4
4
1
8
2
2-Cl
16
8
3-Cl
4-Cl
8
2,4-Cl2
2-Br
3-Br
8
4g
4h
16
8
5

4i
4-Br
4-CH3
2,4-(CH3)2
4-OCH3
2-F
—
—
—
—
—
—
—
—
—
—
—
H
2
4
4
4
4
4
4
8
2
4
32
8
2
4
2
2
2
2
4j
4k
4l
4
8
8
8
8
8
8
4
8
16
16
64
64
16
64
32
64
64
>64
16
>64
>64
64
>64
>64
32
32
32
2
8
16
32
2
8
8
5a
5b
5c
5d
5e
5f
64
16
64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
2
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
>64
>64
64
4-F
32
64
64
64
2-Br
64
>64
32
>64
>64
>64
32
>64
>64
>64
32
3-Br
64
64
>64
16
>64
>64
32
>64
>64
4
4-Br
>64
8
2,4-Cl2
2,4-(CH3)2
2-F
5g
6a
6b
6c
6d
6e
6f
>64
>64
>64
>64
>64
32
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
0.25
n.d.
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
n.d.k
n.d.
1
2-Cl
H
2-Br
H
>64
>64
32
2,4-(CH3)2
2-Cl
H
3-Cl
3-Cl
3-Cl
3-Cl
32
32
6g
4-Cl
32
32
32
32
Gatifloxacin
Moxifloxcin
Fluconazole
0.25
0.25
n.d.
4
2
16
2
2
2
128
n.d.
1
1
n.d.
n.d.
n.d.
n.d.
n.d.
a The antibacterial tests was carried out three times, and the average values were taken as the MICs.
b Staphylococcus aureus 4220. c Staphylococcus aureus 503. d Staphylococcus aureus 209.
e Streptococcus mutans 3065. f Escherichia coli 1924. g Escherichia coli 1356. h Salmonella typhimurium 1926.
i Pseudomonas aeruginosa 2742. j Candida albicans 7535. k n.d.: Not determined.
Table 2. Inhibitory activities (MICa, µg/mL) of compounds 4a–l, 5a–g and 6a–g against the
clinical isolates of multidrug-resistant Gram-positive bacterial strains.
Multidrug-resistant Gram-positive strains
Compd
R
R1
MRSA
QRSA
3167b
3506c
3505d
3519e
8
4a
4b
4c
4d
4e
4f
2-F
—
—
—
—
—
—
—
—
—
—
—
8
8
8
4
4
8
8
2
8
8
8
4
4
8
2
2
8
8
2
2
2
4
8
8
8
4
4
8
8
2
4
4
8
4-F
8
2-Cl
16
8
3-Cl
4-Cl
8
2,4-Cl2
2-Br
16
16
4
4g
4h
4i
3-Br
4-Br
16
4
4j
4-CH3
2,4-(CH3)2
4k
8
6

4l
4-OCH3
2-F
—
—
16
64
8
64
32
>64
32
64
16
64
64
64
>64
>64
32
32
32
2
16
64
16
64
5a
5b
5c
5d
5e
5f
4-F
—
32
32
32
2-Br
—
>64
32
>64
64
>64
64
3-Br
—
4-Br
—
>64
16
>64
16
64
2,4-Cl2
2,4-(CH3)2
2-F
—
16
5g
6a
6b
6c
6d
6e
6f
—
64
>64
>64
64
>64
>64
64
H
64
2-Cl
H
64
2-Br
H
>64
>64
32
>64
>64
32
>64
>64
32
2,4-(CH3)2
2-Cl
H
3-Cl
3-Cl
3-Cl
3-Cl
32
32
32
6g
4-Cl
32
32
32
Gatifloxacin
Norfloxacin
Oxacillin
2
8
4
8
4
>64
>64
1
>64
>64
1
a The antibacterial tests were carried out three times, and the average values were taken as the MICs.
b Methicillin-resistant Staphylococcus aureus 3167. c Methicillin-resistant Staphylococcus aureus 3506.
d Quinolone-resistant Staphylococcus aureus 3505. e Quinolone-resistant Staphylococcus aureus 3519.
The in vitro antibacterial and antifungal activities of the synthesized compounds
are shown in Tables 1 and 2. Most of the synthesized compounds showed potent
inhibitory activities against the different bacteria and the one fungus tested in the
current study with MICs ranging from 1 to 64 µg/mL. Almost all of the compounds in
series 4 exhibited potent antibacterial activity with MICs ranging from 1 to 16 µg/mL,
except for 4i and 4l against the E. coli 1356 strain (MIC = 32 µg/mL). Compounds 4f
and 4h exhibited the highest activity of all the compounds synthesized in series 4
against S. typhimurium 1926 with an MIC value of 1 µg/mL, making them 2-fold
more potent than gatifloxacin and equipotent to moxifloxacin. Against the
Gram-positive strains (S. aureus 503 and S. aureus 209) and Gram-negative E. coli
1924, these two compounds showed equipotent or more potent to the standard drugs
gatifloxacin and moxifloxacin with an MIC value of 2 µg/mL. Against the fungus C.
albicans 7535, compound 4f displayed the strongest potency of all of the compounds
synthesized in series 4 with an MIC value of 1 µg/mL, which was equal to that of
fluconazole. Notably, we have shown for the first time the development of chalcone
7

derivatives exhibiting good antibacterial activity against four Gram-negative bacteria,
especially S. typhimurium 1926 and P. aeruginosa 2742, with MICs ranging from 1 to
16 µg/mL (except 4i and 4l). These compounds also exhibited antifungal activity
against C. albicans 7535 with MICs ranging from 1 to 8 µg/mL, making them more
potent than our previously reported chalcone derivatives.26 The position of the
substituents on the phenyl ring of compounds 4a–l had a pronounced effect on their
activities, which varied according to the following orders: 3-Br > 4-Br > 2-Br for the
bromo-substituted compounds; 2,4-Cl2 > 4-Cl > 3-Cl > 2-Cl for the chloro-substituted
compounds; and 4-CH3 > 2,4-(CH3)2 > 4-OCH3 for compounds bearing an
electron-donating group. Compounds in series 5 and 6 generally showed weak activity
against all of the strains tested in the current study with MICs ranging from 16 to 64
µg/mL. Notably, compound 5f bearing a 2,4-Cl2 substituted phenyl ring showed
moderate activity against all of the strains tested in this study with MICs ranging from
4 to 32 µg/mL, except for the Gram-negative strains S. typhimurium 1926 and P.
aeruginosa 2742. For the compounds in series 6, only those bearing a Cl substituent
on the phenyl ring of their benzoyl hydrazone (6e, 6f, 6g) showed moderate
antimicrobial activity.
As shown in Table 2, most of the compounds exhibited good inhibitory activity
towards the multidrug-resistant Gram-positive bacterial strains (MRSA 3167 and 3506,
QRSA 3505 and 3519) with MIC values in the range of 2–64 µg/mL. The
bromo-substituted compound 4h exhibited the most potent activity against the MRSA
and QRSA strains with MIC values of 2–4 µg/mL, making more potent or equipotent
to gatifloxacin and norfloxacin (MICs of 8 and 4 µg/mL against MRSA 3167 and
3056).
In general, the antimicrobial activity results revealed that the compounds in
series 4 were much more potent than the compounds in the other two series,
indicating that the aminoguanidine moiety was critical to the activity of these chalcone
derivatives. Furthermore, our previously reported chalcone derivatives did not show
any activity against the Gram-negative bacteria tested in this study, and the
introduction of an aminoguanidine moiety to these chalcone resulted in moderate to
8

good levels of activity against several Gram-negative strains of bacteria. It is also
noteworthy that compounds 4f and 5f, bearing a 2,4-Cl2 substituted phenyl ring,
showed excellent antimicrobial activities with MICs ranging from 1 to 8 µg/mL and 4
to 32 µg/mL, respectively, against most of the bacteria strains and the fungus tested in
this study (except Salmonella typhimurium 1926 and Pseudomonas aeruginosa 2742).
These results therefore provide further evidence to suggest that the 2,4-Cl2 substituted
phenyl ring plays a critical role in the activity of these compounds, which is consistent
with the results obtained for a previously reported series of rhodanine derivatives. 30
Table 3. Anti-inflammatory activities of compounds 4a–l, 5a–g and 6a–g following i.p.
administration.
Number
of mice
Edema mean
S.D. (mg)
Inhibition
rate (%)
R
R1
Dose (mg/kg)
DMSO
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
H
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10.60 1.35
1.12 0.07***
1.33 0.10***
0.88 0.28***
0.98 0.30***
1.68 0.53***
2.05 0.65***
4.61 0.65***
0.80 0.21***
2.30 1.19***
5.33 1.57***
3.30 1.40***
2.36 0.66***
3.80 0.68***
4.53 1.00***
5.16 1.20***
5.18 1.36**
5.43 1.50**
9.12 1.50
4.70 0.72***
1.72 0.13***
4.40 1.06***
12.79 0.22
11.42 0.94
11.73 0.68
10.58 0.40
6.04 1.00*
—
Indometacin
—
89.38
87.36
91.66
90.70
84.12
80.66
56.44
92.45
78.30
49.68
68.86
77.66
64.15
57.23
51.25
51.13
48.74
13.96
55.66
83.77
58.49
—
Ibuprofen
—
4a
2-F
4b
4c
4d
4e
4f
4-F
2-Cl
3-Cl
4-Cl
2,4-Cl2
2-Br
4g
4h
4i
3-Br
4-Br
4j
4-CH3
2,4-(CH3)2
4-OCH3
2-F
4k
4l
5a
5b
5c
5d
5e
5f
4-F
2-Br
3-Br
4-Br
2,4-Cl2
2,4-(CH3)2
2-F
5g
6a
6b
6c
6d
6e
2-Cl
H
—
2-Br
H
—
2,4-(CH3)2
2-Cl
H
0.18
3-Cl
43.01
9

6f
3-Cl
4-Cl
3-Cl
3-Cl
100
100
10
10
11.43 1.08
10.69 0.62
—
—
6g
*: p < 0.05, **: p < 0.01, ***: p < 0.001 compared with a vehicle group.
—: no anti-inflammatory activity
The anti-inflammatory effects of the synthesized compounds were also evaluated
in this study, and the results are shown in Table 3. Dimethyl sulfoxide was used as the
vehicle for the primary screening of the synthesized compounds, using indomethacin
and ibuprofen as suitable reference drugs. Compounds were screened in a
xylene-induced ear-edema model in mice, where the anti-inflammatory activity was
assessed based on the ability of a test compounds to prevent edema.31 The results
revealed that most of the synthesized compounds showed pronounced
anti-inflammatory effects at 100 mg/kg. It is noteworthy that compound 4f exhibited
the most potent anti-inflammatory activity of all the compounds with an activity of
92.45%, which was higher than those of ibuprofen (87.36%) and indomethacin
(89.38%). Compounds 4a, 4b and 4f displayed slightly improved activity compared
with the reference drugs. Disappointingly, compounds 6a, 6b, 6c, 6f and 6g did not
exhibit any anti-inflammatory activity at the same dose. The anti-inflammatory
activities of these three series of compounds were therefore determined to be in the
order of series 4 > series 5 > series 6, which indicated that the aminoguanidine moiety
was making a bigger contribution to the biological activity than the acylhydrazone
moiety.
Table 4. Anti-inflammatory activity of compound 4f administered orally at different times before
xylene application.
Inhibition (%)
Time (h)
Dose (mg/kg)
Number of mice
4f
Indometacin
1
2
100
100
100
100
100
100
10
10
10
10
10
10
20.69
34.19**
26.42
16.91
11.28
5.52
11.96
30.94**
18.65
3
4
15.63
5
10.28
24
5.01
**: p < 0.01 compared with a vehicle group.
10

Table 5. Anti-inflammatory activity of compound 4f administered orally at different doses.
Inhibition (%)
Time (h)
Dose (mg/kg)
Number of mice
4f
Indometacin
33.32
2
2
2
100
50
10
10
10
43.45
33.70
17.64*
22.76
4.85*
25
*: 0.01 < p < 0.05 compared with a vehicle group.
Based on their promising anti-inflammatory activity, compound 4f was chosen
for further evaluation. A dose of 100 mg/kg was orally administered at different time
intervals (1, 2, 3, 4, 5 and 24 h) for xylene application. As shown in Table 4, the
activity profile of compound 4f was consistent with that of indomethacin. Notably, the
activity of compound 4f (34.19%) reached its peak at 2 h and was found to be more
potent than indomethacin at this time point (30.94%). The effect of the dosage on the
activity of compound 4f was also evaluated at concentrations of 25, 50 and 100 mg/kg
at 2 h after oral administration (Table 5). The results showed a maximal effect with an
ear inflammation inhibition rate of 43.45% at 100 mg/kg.
Table 6. Cytotoxic activity (IC50a, µg/mL) of compound 4f against human cell lines.
Substituents
R
In vitro cytotoxicity IC50a (µg/mL)
Compound
HeLab
8.7
Hep3Bc
L02d
18.1
4f
2,4-Cl2
15.3
a IC50 is the concentration required to inhibit the cell growth by 50%. Data represent the average of three
independent experiments running in triplicate. Variation was generally between 5–10%.
b Human cervical cancer cells.
c Human liver cancer cells.
d Human normal hepatic cells.
To determine whether the antibacterial and antifungal activities of the
synthesized compounds were selectively toxic towards bacteria and the one fungus,
we evaluated the cytotoxicity of the compound 4f using a standard technique.32 As
shown in Table 6, compound 4f gave IC50 values of 8.7, 15.3 and 18.1 µg/mL against
HeLa, Hep3B and L02 cells, respectively, which were slightly higher than its MIC
values. The result showed the compound 4f does not have much selectivity. Hence it
is difficult to deduce that the promising antibacterial activity of these compounds is
11

not due to their toxicity, but some unknown mechanism of action.33
The mechanisms of action responsible for the antimicrobial and
anti-inflammatory activities of these compounds remain unknown. Lee et al reported
the chalcone derivative cardamomin (2′,4′-dihydroxy-6′-methoxychalcone) as an
inhibitor of NF-κB activation and demonstrated the anti-inflammatory activity of this
compound by blocking the nuclear factor-κB signaling pathway.34 This result therefore
suggests that a similar mechanism could be involved for our synthesized compounds.
Further study towards determining the mechanisms of action of these compounds is
currently underway in our laboratory.
In summary, we have synthesized three series of chalcone derivatives containing
an aminoguanidine or acylhydrazone moiety and evaluated their antibacterial,
antifungal and anti-inflammatory activities. Compounds 4f and 4h showed the highest
levels of activity against the Gram-negative strain S. typhimurium 1926 and the
fungus Candida albicans 7535 with an MIC value of 1 µg/mL, making them 1- and
2-fold more potent than the standard drugs. Furthermore, compound 4f showed the
most potent inhibitory activity of all of the synthesized compounds towards ear
inflammation (92.45%), which was higher than those of ibuprofen (87.36%) and
indomethacin (89.38%) at 100 mg/kg (i.p.). These results therefore suggest that
chalcone derivative 4f could potentially be used to develop potent antibacterial,
antifungal and anti-inflammatory agents for the clinical treatment of numerous
diseases. The mechanisms of action of these compounds are currently being
elucidated in our laboratories and will be reported in due course.
Acknowledgment
This work was supported by the National Natural Science Foundation of China
(Grant numbers 81260468, 81460524 and 81060257).
Supplementary data
Supplementary data associated with this article can be found in the online
version.
12

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28. Preparation of 4f: Terephthalaldehyde 2 (6 mmol) with 2,4-Dichloroacetophenone
(5 mmol) in refluxing ethanol (50 mL) at 23 °C in the presence of NaOH (0.4g, 10
mmol)
for
3–4
h
afforded
(E)-4-(3-(2,4-dichlorophenyl)-3-oxoprop-1-en-1-yl)benzaldehyde in 60%–70%
yields,26,27 which reacted with aminoguanidine hicarbonate (3 mmol) in refluxing
ethanol (20 mL) in the presence of 5 drops of concentrated hydrochloric acid at
60–70 °C for 8–12 h. The solution was evaporated to dryness under reduced
pressure, and the residue was purified by silica gel column chromatography with
dichloromethane: methanol (50:1) to afforded yellow solid 4f. Yield 71%; m.p.
o
203–205 C; IR (KBr cm-1): 3373 (NH2), 3271 (C=NH), 3174(NH), 1680 (C=O),
1597 (C=N). 1H NMR (DMSO-d6, 300 MHz, ppm) δ 11.95 (br.s, 1H, NH), 8.19 (s,
1H, CH=N), 7.95-7.80 (m, 7H, CH, NH, NH2 and Ar-H), 7.66-7.58 (m, 2H, CH
and Ar-H), 7.49-7.32 (m, 3H, Ar-H). 13C NMR (DMSO–d6, 75 MHz): δ 192.88,
14

156.01, 146.43, 146.15, 137.73, 136.32, 136.20, 136.12, 131.69, 131.21, 130.15,
129.75, 129.75, 128.48, 128.48, 128.14, 127.29. HRMS (MALDI) calcd for
C17H15Cl2N4O (M + H) +: 361.0621, found: 361.0624.
29. Evaluation of anti-bacterial activity in vitro: The micro-organisms used in the
present study were S. aureus 4220, S. aureus 209, S. aureus 503, Streptococcus
mutans 3065, Escherichia coli 1924, Escherichia coli 1356, Pseudomonas
aeruginosa 2742, Salmonella typhimurium 1926 and one fungus (Candida
albicans 7535). The strains of multidrug-resistant clinical isolates were
methicillin-resistant Staphylococcus aureus (MRSA 3167 and 3506) and
quinolone-resistant Staphylococcus aureus (QRSA 3505 and 3519). Clinical
isolates were collected from various patients hospitalized in several clinics. The in
vitro antimicrobial activity was evaluated using the minimum inhibitory
concentration (MIC) with different strains. Gatifloxacin, moxifloxacin,
fluconazole, norfloxacin, and oxacillin were used as positive controls. The
bacteria were grown to mid-log phase in Mueller-Hinton broth and diluted
1000-fold in the same medium. Stock solutions of the test compounds in dimethyl
sulfoxide were prepared and then poured into 96-well plates. The final
concentration of 0.5-64 µg/mL underwent a twofold serial dilution. The
microbacteria were suspended and contained approximately 105 CFU/mL. These
were applied to 96-well plates with a serial dilution and incubated at 37 °C for 24
h. The microbacterial growth was measured from the absorption at 630 nm. This
was done using a microtiter, enzyme-linked immunosorbent assay (ELISA) reader.
All experiments were carried out in triplicate.
30. Song M. X.; Zheng, C. J.; Deng X. Q.; Wei Z. Y.; Piao, H. R. Med. Chem. 2014, 4,
441.
31. Anti-inflammatory assay: Assay in the xylene-induced ear edema method via the
intraperitoneal route: The anti-inflammatory activity was evaluated by an in vivo
inhibition assay by monitoring xylene-induced ear edema in mice. In the primary
screening, all tested compounds, ibuprofen and indomethacin were freshly
prepared (dissolved with DMSO) prior to administered i.p. at a dose of 100 mg/kg
15

to mice and at a concentration of 0.1 mL/20 g body weight. Control mice received
the vehicle only (DMSO, 0.1 mL/20 g of body weight). Thirty minutes after
administration i.p., animals were used in the xylene-induced ear edema test, 20 μL
xylene was applied to the surface of the right ear of each mouse by a micropipette.
After keeping them from struggling for 30 min, a cylindrical plug (diameter, 7 mm)
was excised from each of the treated and untreated ears. Edema was quantified by
the difference in weight between the two plugs. Anti-inflammatory activity was
expressed as percent reduction in edema compared with the DMSO-administered
control group. The NSAID ibuprofen and indomethacin were tested in parallel as
reference.
Assay in the xylene-induced ear edema method via the oral route: In the latter
evaluation, tested compounds and indometacin were homogenized with 0.5%
sodium carboxymethylcellulose (CMC-Na) and administered via the oral route to
mice at a concentration of 0.2 mL/20 g mice weight. Control mice received the
vehicle only (0.5% CMC-Na, 0.2 mL/20 g). To explore the peak activity of the
compound, edema was quantified at different intervals (1, 2, 3, 4, 5, and 24h).
Compounds 4f and indomethacin homogenized with 0.5% CMC-Na were
administered orally to mice (lower doses of 50 mg/kg and 25 mg/kg and 0.2 mL/20
g mice body weight). Control mice received 0.5% CMC-Na (0.2 mL/20 g body
weight) and edema quantified at the peak interval of 3 h. Edema was quantified by
the difference in weight between the two plugs. Edema values, expressed as mean
standard deviation, were compared statistically using one-way-ANOVA followed
by Dunnet’s test. A level of p < 0.05 was adopted as the test of significance.
32. Cytotoxicity on human cancer cells: The cytotoxicity test of selected compounds
was measured through the colorimetric MTT assay. Human cervical cancer cells
(HeLa) and liver cancer cells (Hep3B) suspension in DMEM medium
supplemented with 10% FBS and antimycotic was added in 96-well microplates at
1.8×104 cells/well. A variety of concentrations of the test compounds (200, 100,
50, 25, 12.5, 6.25, 3.125, 1.625 µM/L) dissolved by distilled 10% DMSO was
added to each well. Incubation for 24h at 37℃ under 5% CO2, 2.5mg/mL of MTT
16

solution was added to each well. Further the plate was incubated for 4h. Then, the
medium was removed and the resulting formazan crystals were dissolved with 100
µL DMSO. After shaking 10 min, the optical density was measured at 570 nm
using a microtiter ELISA reader. The selected compounds were used as positive
control, whereas untreated cells were used as negative controls. The IC50 values
were defined as the concentrations inhibiting 50% of cell growth. All experiments
were performed in triplicate.
33. Song, M. X.; Deng, X. Q.; Wei, Z. Y.; Zheng, C. J.; Wu, Y.; An, C. S.; Piao, H. R.
Iran. J. Pharm. Res. 2015, 14, 89.
34. Lee, J. H.; Jung, H. S.; Giang, P. M.; Jin, X. J.; Lee, S.; Son, P. T.; Lee, D.; Hong,
Y. S.; Lee, K.; Lee J. J. J. Pharmacol. Exp. Ther. 2006, 316, 271.
17

Synthesis and biological evaluation of chalcone derivatives
containing aminoguanidine or acylhydrazone moieties
Zhi-Yu Wei a, Ke-Qiang Chi b, Zhan-Kui Yu a, Hong-Yan Liu a, Liang-Peng Sun a,
Chang-Ji Zheng a,*, Hu-Ri Piao a,*
a Key Laboratory of Natural Resources and Functional Molecules of the Changbai
Mountain, Affiliated Ministry of Education; Yanbian University College of Pharmacy,
Yanji, Jilin Province, 133002, PR China
b Medical College of Dalian University, Dalian, Liaoning Province, 116622, PR China
H
N
NH2
NH
N
R
O
4a-l
MIC = 2 g/mL (E. coli)
MIC = 1 g/mL (C. albicans)
N
H
N
H
N
R1
N
N
O
O
R
R
O
O
5a-g
6a-g
Three novel series of chalcone derivatives containing aminoguanidine or
acylhydrazone moieties were designed, synthesized, characterized and evaluated for
their antibacterial activity, antifungal activity and anti-inflammatory activity.
18