Synthesis and biological evaluation of 1,3-diaryl pyrazole derivatives as potential antibacterial and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2015.10.028

Three series of 1,3-diaryl pyrazole derivatives bearing aminoguanidine or furan-2-carbohydrazide moieties have been synthesized, characterized and evaluated for antibacterial and anti-inflammatory activities. Most of the synthesized compounds showed potent inhibition of several Gram-positive bacterial strains (including multidrug-resistant clinical isolates) and Gram-negative bacterial strains with minimum inhibitory concentration values in the range of 1-64 μg/mL. Compounds 6g, 6l and 7l presented the most potent inhibitory activity against Gram-positive bacteria (e.g. Staphylococcus aureus 4220), Gram-negative bacteria (e.g. Escherichia coli 1924) and the fungus, Candida albicans 7535, with minimum inhibitory concentration values of 1 or 2 μg/mL. Compared with previous studies, these compounds exhibited a broad spectrum of inhibitory activity. Furthermore, compound 7l showed the greatest anti-inflammatory activity (93.59% inhibition, 30 min after intraperitoneal administration), which was more potent than the reference drugs ibuprofen and indomethacin.

Full text of DOI:10.1016/j.bmcl.2015.10.028

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 1,3-diaryl pyrazole derivatives
as potential antibacterial and anti-inflammatory agents
⇑
⇑
Ya-Ru Li, Chao Li, Jia-Chun Liu, Meng Guo, Tian-Yi Zhang, Liang-Peng Sun, Chang-Ji Zheng , Hu-Ri Piao
Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, Yanbian University, College of Pharmacy, Yanji 133002,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of 1,3-diaryl pyrazole derivatives bearing aminoguanidine or furan-2-carbohydrazide moi-
eties have been synthesized, characterized and evaluated for antibacterial and anti-inflammatory activ-
ities. Most of the synthesized compounds showed potent inhibition of several Gram-positive bacterial
strains (including multidrug-resistant clinical isolates) and Gram-negative bacterial strains with mini-
Received 11 July 2015
Revised 8 October 2015
Accepted 10 October 2015
Available online xxxx
mum inhibitory concentration values in the range of 1–64 lg/mL. Compounds 6g, 6l and 7l presented
the most potent inhibitory activity against Gram-positive bacteria (e.g. Staphylococcus aureus 4220),
Gram-negative bacteria (e.g. Escherichia coli 1924) and the fungus, Candida albicans 7535, with minimum
inhibitory concentration values of 1 or 2 lg/mL. Compared with previous studies, these compounds
exhibited a broad spectrum of inhibitory activity. Furthermore, compound 7l showed the greatest anti-
inflammatory activity (93.59% inhibition, 30 min after intraperitoneal administration), which was more
potent than the reference drugs ibuprofen and indomethacin.
Keywords:
Pyrazole
Aminoguanidine
Furan
Antibacterial activity
Anti-inflammatory activity
Ó 2015 Elsevier Ltd. All rights reserved.
The incidence of bacterial infection has increased rapidly during
the past two decades.1 Furthermore, many drug-resistant patho-
gens have emerged in recent years because of the increasing use
or abuse of antibacterial agents for all kinds of human infectious
diseases.1–4 In addition to bacterial infection, many factors can lead
to inflammation, such as biological agents, physical agents, chem-
ical injuries and allergic reactions. Such conditions may lead to
bacteremia, toxemia, septicemia and pyemia. As a result, the devel-
opment of novel antibacterial and anti-inflammatory agents is cru-
cial for ongoing effective therapeutic intervention.5–8
Pyrazoles occupy a distinct niche in heterocyclic chemistry and
represent a key motif in medicinal chemistry because of their capa-
bility to exhibit an array of bioactivities such as antimicrobial,9,10
anticancer,11 anti-inflammatory,12 antidepressant,13 anticonvul-
sant,14 antipyretic15 and selective enzyme inhibitory16 activities.
In recent years, the guanidinium derivatives have been infre-
quently investigated as pharmaceutical antimicrobial agents.17 In
our previous work, we found that several pyrazole derivatives
showed moderate to good activity against Gram-positive strains
(including multidrug-resistant clinical isolates).5,9 One of these
derivatives, compound A, exhibited inhibitory activity with a
minimum inhibitory concentration (MIC) value of 4 lg/mL
(Fig. 1).5 Unfortunately, however, none of these derivatives
displayed any activity against Gram-negative bacteria, even at
64
antibacterial activity against Escherichia coli with an MIC value of
32
g/mL (Fig. 1).18 In this work, as part of our ongoing studies
lg/mL. Dibama et al. reported that compound B showed potent
l
toward the development of novel antibacterial agents, we designed
new hybrid compounds in which the rhodanine moiety of A was
replaced by a guanidine moiety or its isostere furan-2-carbohy-
drazide, simultaneously changing the substituents on the phenyl
ring of the pyrazole, to investigate their effects on activity. Inspired
from existing antibacterial agents bearing nitro groups, such as
chloramphenicol that operates via inhibition of bacterial protein
synthesis after the nitro group is metabolized to a hydroxyamino
group, two nitro groups were introduced into the phenyl ring on
the N1 position of the pyrazole. Thus, three novel series of
1,3-diaryl pyrazole derivatives bearing aminoguanidine or
furan-2-carbohydrazide moieties were designed, synthesized, and
evaluated for their antimicrobial activities. Meanwhile, in light of
the fact that some drugs contain a pyrazole moiety, such as phena-
zone and metamizole,19 these compounds were also evaluated for
their anti-inflammatory activity.
The synthesis of the target compounds is presented in
Scheme 1.22 Hydrazone derivatives (compounds 2 and 4) were pre-
pared by reacting substituted acetophenones with phenylhy-
drazine (or 2,4-dinitrophenylhydrazine) in the presence of glacial
⇑
Corresponding authors. Tel.: +86 (433)243 6015; fax: +86 (433)243 5026
(C.-J.Z.); tel.: +86 (433)243 5003; fax: +86 (433)243 5026 (H.-R.P.).
E-mail addresses: zhengcj@ybu.edu.cn (C.-J. Zheng), piaohr@ybu.edu.cn
(H.-R. Piao).
http://dx.doi.org/10.1016/j.bmcl.2015.10.028
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Li, Y.-R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.028

2
Y.-R. Li et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Cl
H
N
NH
Cl
CF3COOH
O
NH2
COOH
N
N
O
N
S
S
Compound B MIC= 32 µg/mL
Compound A MIC= 4 µg/mL
R
N
N
NH
NH2
N
R1
N
H
6a-p: R1= 2,4-dinitrophenyl
1
7c 7h 7j 7l 7m
,
,
,
,
: R = phenyl
Figure 1. Previously reported compound A, B and the structure-based design of the target compounds.
NH2
HN
NO2
NO2
NH
NO2
N
NO2
NH
c
O
H2CO3
O2N
H2N
R
O2N
O2N
N
H
NH2
b
NO2
a
N
N
N
N
R
NH
NH2
1
R
R
N
CHO
N
H
H
N
2
3
HN
6a-p
NH2
NH2
a
O
O
d
R
NO2
N
N
H
O2N
4
N
N
b
R
O
N
N
O
H
NH
H2CO3
NH2
H2N
N
H
8c, 8f, 8g, 8i, 8l
N
N
N
R
c
N
NH
NH2
N
R
N
CHO
H
5
7c, 7h, 7j, 7l, 7m
R= a: 4-OCH3 b: 3-OCH3 c: 4-CH3 d: 3-CH3 e: 2,4-(CH3)2 f: 2-Br g: 3-Br h: 4-Br
i: 2-F j: 4-F k: 2-Cl l: 3-Cl m:4-Cl n: 2,4-(Cl)2 o: H p: 2-NO2
Scheme 1. Synthetic scheme for the synthesis of compounds 6–8. Reagents and conditions: (a) EtOH, AcOH or AcONa, reflux 5–7 h, (b) POCl3, DMF, 0 °C 0.5 h, 80 °C 6 h, (c)
EtOH, HCl, Reflux 20 h. (d) EtOH, reflux 3 h.
acetic acid in ethanol. Compound 2 (or compound 4) reacted under
Vilsmeier–Haack (DMF–POCl3) conditions and afforded corre-
sponding 4-carboxaldehyde functionalized pyrazole derivatives 3
(or 5). In this reaction, four equivalents of the reagent, instead of
three as described by Pascal Rathelot,20 were necessary to obtain
compounds 3 and 5 in good yield. Compounds 3 or 5 were then
reacted with aminoguanidine bicarbonate in the presence of cat-
alytic amounts of hydrochloric acid in ethanol to provide com-
pounds in series 6 and 7. Compounds in series 8 were prepared
by reacting compounds 3 with furan-2-carbohydrazide in refluxing
ethanol. The structures of the compounds synthesized were con-
firmed by IR, 1H NMR, 13C NMR, and mass spectroscopy.
In vitro anti-bacterial activities of the synthesized compounds
were evaluated using a 96-well microtiter plate and a serial dilu-
tion method to obtain the Minimum Inhibitory Concentration
(MIC) values for different strains (including multidrug-resistant
clinical isolates). Four Gram-positive strains (Staphylococcus aureus
RN 4220, S. aureus KCTC 209, S. aureus KCTC 503, Streptococcus
mutans 3065), five Gram-negative strains (Escherichia coli KCTC
1924, Escherichia coli CCARM 1356, Pseudomonas aeruginosa 2742,
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Y.-R. Li et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Pseudomonas aeruginosa 2004, Salmonella typhimurium 2421) and
one fungus (Candida albicans 7535) were used for the biological
assay. Gatifloxacin, moxifloxacin, norfloxacin, and oxacillin were
used for the antibacterial activity, fluconazole and itraconazole
for the antifungal activity as positive controls. In vivo inflamma-
tory inhibition assay was carried out using xylene-induced ear
edema21 model in mice. Indomethacin and ibuprofen were used
as positive controls.
of 1
(MIC = 1
positive control moxifloxacin (MIC = 2
(MIC = 2 g/mL) against E. coli 1924. Compounds 6c, 6g, 6h and
6l represented a 4-fold increase in potency relative to the standard
drug gatifloxacin (MIC = 16 g/mL) with MIC values of 4 g/mL,
l
g/mL against C. albicans 7535. In particular, compound 7l
g/mL) exhibited more potent activity than the
g/mL) and gatifloxacin
l
l
l
l
l
and 6a, 6b, 6d, 6e, 6j, 6m, 6n and 7c displayed a 16-fold increase
in potency relative to the standard drug moxifloxacin
As indicated in Table 1,23 most of the tested compounds except
for the compounds in series 8 showed potent antibacterial and
antifungal activity against different strains including Gram-posi-
tive strains (Staphylococcus aureus and Streptococcus mutans),
Gram-negative strains (E. coli, Salmonella typhimurium and Pseu-
domonas aeruginosa) and one fungus (Candida albicans 7535), with
(MIC = 128 lg/mL) with MIC values of 8 lg/mL against E. coli
1356. For the P. aeruginosa 2742 and 2004 strains, however,
only compound 7c exhibited good activity with MIC values of
32
l
g/mL and 16
lg/mL, respectively.
As indicated in Table 2,23 all of the synthesized compounds
were also tested for their inhibitory activities against the clinical
isolates of several different multidrug-resistant Gram-positive bac-
terial strains (MRSA CCARM 3167 and 3506, QRSA CCARM 3505 and
3519). The MIC values of the compounds differed greatly, ranging
from 1 to 32 mg/mL. Compounds in series 6 and 7 exhibited more
potent activity than the compounds in series 8. Compounds 6g and
MIC values in the range of 1–64 lg/mL. As a whole, the compounds
in series 6 exhibited more potent anti-bacterial activities against
all microorganisms compared with series 7. Compounds 6c, 6d,
6g, 6h, 6l, 6m, 6n and 7l showed the strongest activity with MIC
values of 2
lg/mL, equivalent to those of the standard drug
moxifloxacin (MIC = 2
against S. aureus KCTC 209 and 503. Compounds 6g, 6h, 6l and 7l
displayed the same inhibitory effect as fluconazole with MIC values
l
g/mL) and gatifloxacin (MIC = 2 g/mL)
l
7l presented the highest activity with MIC values of 1
against MRSA CCARM 3167, and compounds 6g, 6h, 6l and 7l
showed the highest activity with MIC values of 1 g/mL against
lg/mL
l
Table 1
Inhibitory activity (MIC,
lg/mL) of compounds 6a-p, 7c, 7h, 7j, 7l, 7m, 8c, 8f, 8g, 8i and 8l against strains of Gram-positive (Staphylococcus aureus and Streptococcus mutans)
bacteria, Gram-negative (Escherichia coli, Salmonela typhinurium and Pseudomonas aeruginosa) bacteria and Candida albicans 7535
Compound
R
Gram-positive strains
Gram-negative strains
Fungus
C. albicans
7535j
S. aureus
S. Mutans
E. Coli
P. aeruginosa
S. typhimurium
4220a
209b
503c
3065d
1924e
1356f
2742g
2004h
2421i
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
7c
7h
7j
7l
7m
8c
8f
8g
4-OCH3
3-OCH3
4-CH3
3-CH3
2,4-(CH3)2
2-Br
3-Br
4-Br
2-F
4-F
2-Cl
3-Cl
4-Cl
2,4-(Cl)2
H
2-NO2
4-CH3
4-Br
4-F
3-Cl
4-Cl
4-CH3
2-Br
3-Br
2-F
4
4
2
2
2
4
1
2
8
4
4
1
2
2
8
16
2
4
4
2
2
4
8
2
2
8
4
8
2
2
2
8
32
4
4
4
2
2
4
4
2
2
8
4
4
2
2
2
8
16
4
4
16
2
>64
>64
>64
>64
>64
>64
4
8
8
4
4
4
16
4
2
16
8
4
4
2
2
2
4
2
2
8
4
4
2
2
2
8
16
2
8
8
4
8
8
16
4
4
16
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
8
8
4
8
8
16
4
4
16
8
2
2
2
2
2
2
1
1
4
2
2
1
2
2
4
8
2
4
8
1
>64
>64
>64
>64
>64
>64
0.50
0.50
1
16
4
4
16
4
8
8
4
4
8
4
8
16
32
8
>64
>64
8
>64
>64
>64
>64
>64
>64
0.50
0.25
n.d.
n.d.
16
32
8
16
32
8
>64
>64
8
>64
>64
>64
>64
>64
>64
0.50
0.50
n.d.
n.d.
4
8
2
8
8
2
8
8
1
>64
>64
16
>64
>64
>64
>64
>64
>64
16
128
n.d.
n.d.
16
>64
>64
>64
>64
>64
0.25
0.25
n.d.
n.d.
>64
>64
>64
>64
>64
>64
2
2
n.d.
n.d.
>64
>64
>64
>64
>64
>64
2
2
n.d.
n.d.
8i
8l
3-Cl
—
—
—
Gatifloxacin
Moxifloxacin
Fluconazole
Itraconazole
2
n.d.
n.d.
1
n.d.
n.d.
2
n.d.
n.d.
—
0.06
n.d.: not determined.
S. aureus RN 4220: A genotype of S. aureus. KCTC (Korean Collection for Type Cultures). CCARM (Culture Collection Antimicrobial Resistant Microbes).
a
Staphylococcus aureus RN 4220.
Staphylococcus aureus 209.
Staphylococcus aureus 503.
Streptococcus mutans 3065.
Escherichia coli KCTC 1924.
Escherichia coli CCARM 1356.
Pseudomonas aeruginosa 2742.
Pseudomonas aeruginosa 2004.
b
c
d
e
f
g
h
i
Salmonella typhimurium 2421.
Candida albicans 7535.
j
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Y.-R. Li et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2
Inhibitory activity (MIC,
l
g/mL) of compounds 6a–p, 7c, 7h, 7j, 7l, 7m, 8c, 8f, 8g, 8i and 8l against clinical isolates of multidrug-resistant Gram-positive strains
Compound
R
Multidrug-resistant Gram-positive strains
MRSA
QRSA
3167a
3506b
3505c
3519d
6a
6b
6c
6d
6e
6f
6g
6h
4-OCH3
3-OCH3
4-CH3
3-CH3
2,4-(CH3)2
2-Br
3-Br
4-Br
2-F
4
4
2
2
2
4
1
2
2
2
2
2
2
4
1
1
4
4
2
2
2
4
2
2
4
4
2
4
4
8
2
2
6i
8
4
8
8
6j
4-F
4
4
4
4
6k
2-Cl
4
4
4
8
6l
3-Cl
2
1
2
2
6m
4-Cl
2
2
2
2
6n
6o
2,4-(Cl)2
H
2
8
2
8
2
8
2
8
6p
7c
7h
7j
2-NO2
4-CH3
4-Br
4-F
16
2
8
8
1
8
2
4
8
16
4
16
16
2
32
4
16
16
2
7l
3-Cl
1
7m
8c
8f
8g
8i
8l
Gatifloxacin
Moxifloxacin
Norfloxacin
Oxacillin
4-Cl
>64
>64
>64
>64
>64
>64
2
1
8
>64
>64
>64
>64
>64
>64
>64
2
1
4
>64
>64
>64
>64
>64
>64
>64
8
4
>64
1
>64
>64
>64
>64
>64
>64
4
4
>64
1
4-CH3
2-Br
3-Br
2-F
3-Cl
—
—
—
—
a
Methicillin-resistant S. Aureus CCARM 3167.
Methicillin-resistant S. Aureus CCARM 3506.
Quinolone-resistant S. Aureus CCARM 3505.
Quinolone-resistant S. Aureus CCARM 3519.
b
c
d
Table 3
Anti-inflammatory activity of compounds 6a–p, 7c, 7h, 7j, 7l, 7m, 8c, 8f, 8g, 8i and 8l administrated ip
R
Dose (mg/kg)
Number of mice
Edema mean SD (mg)
Inhibition rate (%)
DMSO
Indometacin
—
—
—
—
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
10
12.99 0.71
7.11 0.50⁄⁄⁄
9.15 0.58⁄
—
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
45.23
29.56
86.53
67.28
87.43
76.90
79.60
78.06
81.65
69.59
87.17
61.89
84.60
82.42
71.52
61.89
86.91
58.30
86.53
42.78
61.38
93.59
45.96
35.95
17.47
25.32
28.02
26.23
Ibuprofen
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
7c
7h
7j
7l
7m
8c
8f
8g
8i
8l
4-OCH3
3-OCH3
4-CH3
3-CH3
2,4-(CH3)2
2-Br
3-Br
4-Br
2-F
4-F
2-Cl
3-Cl
4-Cl
2,4-(Cl)2
H
2-NO2
4-CH3
4-Br
4-F
3-Cl
4-Cl
4-CH3
2-Br
3-Br
2-F
1.75 0.43⁄⁄⁄
4.25 1.95⁄⁄⁄
1.63 0.30⁄⁄⁄
3.00 0.74⁄⁄⁄
2.65 0.65⁄⁄⁄
2.85 0.47⁄⁄⁄
2.38 1.04⁄⁄⁄
3.95 0.67⁄⁄⁄
1.67 0.60⁄⁄⁄
4.95 1.62⁄⁄⁄
2.00 0.32⁄⁄⁄
2.28 0.36⁄⁄⁄
3.70 0.69⁄⁄⁄
4.95 2.11⁄⁄⁄
1.70 0.48⁄⁄⁄
5.42 0.55⁄⁄⁄
1.75 0.61⁄⁄⁄
7.43 1.11⁄⁄⁄
5.17 1.15⁄⁄⁄
0.83 0.26⁄⁄⁄
7.02 0.83⁄⁄⁄
8.32 1.85⁄
10.72 0.97
9.70 1.45
9.35 1.44
3-Cl
9.58 1.04
*: 0.01 < p < 0.05, ***: p < 0.001 compared with vehicle group.
—: no anti-inflammatory activity.
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Y.-R. Li et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 4
anti-inflammatory effects at 50 mg/kg administered via the
intraperitoneal route, with inhibition ranging from 58.30% to
93.59%. It is worth noting that compound 7l exhibited the stron-
gest inflammatory inhibition at 93.59%, higher than ibuprofen
(29.56%) and indomethacin (45.23%). Compounds 7h, 7m and 8c
displayed slightly improved activity compared with the reference
drugs. Compounds 8f, 8g, 8i and 8l, however, did not exhibit any
anti-inflammatory activity at the same dosage, indicating that
the aminoguanidine moiety was more beneficial to biological
activity than the furan-2-carbohydrazide moiety.
Anti-inflammatory activity of compounds 7l administered orally at different times
before xylene application
Time(h)
Dose (mg/kg)
Inhibition (%)
Indometacin
7l
1
2
3
4
5
50
50
50
50
50
50
29.69
9.93
23.02
23.60⁄⁄⁄
41.43
34.89⁄⁄
26.87
—
50.86⁄⁄⁄
4.39
35.18
—
24
In view of its considerable anti-inflammatory activity, com-
pound 7l was chosen for further evaluation. A dose of 50 mg/kg
was administered via the oral route at different intervals (1, 2, 3,
4, 5 and 24 h) for xylene application. As shown in Table 4,24 the
activity profile of compound 7l was distinct from that of indo-
methacin, for which the activity showed no regularity as the inter-
val lengthened. The activity of compound 7l peaked at 3 h and
exhibited more potent activity than indomethacin. In addition,
the effect of dosage on the activity of compound 7l was also tested
at concentrations of 12.5, 25 and 50 mg/kg, 3 h after oral adminis-
tration, and showed a maximal effect with an ear inflammation
inhibition rate of 56.67% at 12.5 mg/kg. Whereas lower activity
was observed at 25 mg/kg administered 3 h after xylene applica-
tion (Table 5).24
In conclusion, based on our previous work, three novel series of
1,3-diaryl pyrazole derivatives bearing aminoguanidine or furan-2-
carbohydrazide moieties were synthesized, characterized, and
evaluated for antibacterial (including Gram-positive and Gram-
negative bacterial strains) and anti-inflammatory activities. The
results showed that most compounds exhibited moderate to good
levels of antibacterial and anti-inflammatory activities. In particu-
lar, compounds 6g, 6l and 7l exhibited the greatest antibacterial
activities against Gram-positive bacterial strains (S. aureus RN
4220, S. aureus KCTC 209, S. aureus KCTC 503, S. mutans 3065, MRSA
CCARM 3167 and 3506, QRSA CCARM 3505 and 3519), and Gram-
negative strains (E. coli KCTC 1924, E. coli CCARM 1356, S. typhimur-
ium 2421), and antifungal activity (C. albicans 7535) with MIC val-
**: 0.001 < p < 0.01, ***p < 0.001 compared with vehicle group.
—: no anti-inflammatory activity.
MRSA CCARM 3506, showed the same potency as moxifloxacin
(MIC = 1 g/mL), 4 to 8-fold more potent activity relative to the
standard drug norfloxacin (MIC = 4–8 g/mL), and 64-fold more
potent activity relative to oxacillin (MIC > 64 g/mL). Compounds
6c, 6g, 6h, 6l-n and 7l exhibited 2 to 4-fold more potent activity
relative to gatifloxacin and moxifloxacin (MIC = 4–8 g/mL) with
MIC values of 2 g/mL against QRSA CCARM 3505 and 3519, but
weaker than that of oxacillin (MIC = 1 g/mL). Regretfully, com-
l
l
l
l
l
l
pounds in series 8 did not exhibit any inhibitory activity against
MRSA CCARM (3167 and 3506) and QRSA CCARM (3505 and 3519)
strains.
Analysis of these results revealed several structure–activity
relationships. First, a comparison of the activities across the three
different series of compounds revealed a general order of antibac-
terial activity of 6 > 7 > 8, which indicated that the aminoguanidine
moiety was critical for the anti-bacterial activity of pyrazole
derivatives. Second, none of the pyrazole derivatives previously
reported by our group showed any activity against the Gram-neg-
ative bacteria E. coli 1924 and 1356, and P. aeruginosa 2742 and
2004.5,9 However, the introduction of an aminoguanidine moiety
resulted in moderate to good levels of inhibition against the
Gram-negative strains. In series 7, only the compound 7l exhibited
an equal potency to those compounds (most of which exhibited
strong activity against all of the microorganisms tested) in series
6, indicating that the 2,4-dinitrophenyl moiety is beneficial for
the activity and the chloro substituent at the 3-position might be
critical for the activity. Third, no clear pattern was found for the
structure/activity relationship between the anti-bacterial activity
and the position and physicochemical properties of different sub-
stituents on the phenyl ring, indicating that the electronic effect
of the substituent on the benzene ring is not critical. As a result,
derivatives 6g, 6l and 7l are promising target compounds to be
investigated further.
ues of 1–8 lg/mL, higher than those observed for the positive
controls. Compound 7c exhibited a good level of broad antimicro-
bial inhibition against all of the strains (including P. aeruginosa
2742 and 2004). Regarding anti-inflammatory activity, compound
7l showed the most potent ear inflammation inhibition rate
(93.59%), which was higher than that of ibuprofen (29.56%) and
indomethacin (45.23%) at 50 mg/kg (ip). These results suggested
that introduction of aminoguanidine into the pyrazole ring was
beneficial to antibacterial activity, providing valuable information
for the design of Gram-negative bacteria inhibitors. The mecha-
nism of action of these compounds remains unknown and further
investigations are currently underway in our laboratories.
All of the synthesized compounds were also evaluated for their
anti-inflammatory activity and the results are shown in Table 3.24
In the primary screening, we used dimethyl sulfoxide as the vehicle
and ibuprofen and indomethacin as the reference drugs. In this
test, compounds were screened in a xylene-induced ear-edema
test in mice, in which anti-inflammatory activity was assessed by
the ability of the test compounds to prevent edema. The data
revealed that most of the test compounds showed significant
Acknowledgment
This work was supported by the National Science Foundation of
China (Grant numbers 81260468, 81060257, and 81460524).
Supplementary data
Table 5
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
028.
Anti-inflammatory activity of compound 7l administered orally at different doses
Time (h)
Dose (mg/kg)
Inhibition (%)
Indometacin
7l
53.66⁄⁄⁄
39.28⁄⁄⁄
56.67⁄⁄⁄
52.69⁄⁄⁄
53.89⁄⁄⁄
56.27⁄⁄⁄
References and notes
3
3
3
50
25
12.5
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Please cite this article in press as: Li, Y.-R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.028

6
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Assay in the xylene-induced ear edema method via the intraperitoneal route:
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mice and at
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test, 20 lL 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 0.4 mL/20 g mice weight. Control mice received
the vehicle only (0.5% CMC-Na, 0.4 mL/20 g). To explore the peak activity of the
compound, edema was quantified at different intervals (1, 2, 3, 4, 5, and 24 h).
Compounds 7l and indomethacin homogenized with 0.5% CMC-Na were
administered orally to mice (lower doses of 25 mg/kg and 12.5 mg/kg and
0.4 mL/20 g mice body weight). Control mice received 0.5% CMC-Na (0.4 mL/
20 g body weight) and edema quantified at the peak interval of 3 h.
22. Preparation of 6l: First, compound 3l (1 mmol) was dissolved in absolute
ethanol (10–15 mL). Second, aminoguanidine bicarbonate (1.1 equiv) and
catalytic amounts of hydrochloric acid were added. Then, the reaction
mixtures were refluxed for 20 h. Finally, the solvent was evaporated under
reduced pressure and the crude product was purified by silica gel column
chromatography (dichloromethane/methanol, 15:1) to afford the desired
compounds as yellow solid. Yield 81.59%; mp 236–238 °C. IR (KBr) cmÀ1
:
3456, 3238, 3120 (NH2, NH), 1676 (C@N), 1548, 1354 (NO2). 1H NMR (DMSO-
d6, 300 MHz, ppm): d 7.59–7.66 (m, 4H, Ar–H), 7.81 (br s, 3H, NH), 8.25 (d, 1H,
J = 9 Hz, Ar–H), 8.28 (s, 1H, pyrazole-H), 8.74 (d, 1H, J = 9 Hz, Ar-H), 8.96 (d, 1H,
J = 3 Hz, Ar-H), 9.28 (s, 1H, CH@N), 11.89 (br s, 1H, NH). 13C NMR (DMSO-d6,
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
75 MHz, ppm):
d
155.67, 152.10, 146.46, 143.14, 138.60, 135.56, 134.13,
p < 0.05 was adopted as the test of significance.
133.40, 132.32, 131.40, 129.66, 128.75, 128.04, 127.30, 125.96, 121.86, 118.71.
MS m/z 429 (M+1).
Please cite this article in press as: Li, Y.-R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.028