Synthesis and biological evaluation of 5-aryloxypyrazole derivatives bearing a rhodanine-3-aromatic acid as potential antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2012.09.107

Three novel series of 5-aryloxypyrazole derivatives have been synthesized and tested for their antibacterial activity. The majority of the synthesized compounds showed potent inhibitory activity against Gram-positive bacteria Staphylococcus aureus 4220, especially against the strains of multidrug-resistant clinical isolates (MRSA3167/3506 and QRSA3505/3519). Among which compounds IIIb, IIIg and IIIm showed the most potent levels of activity (MIC = 1 μg/mL) against the multidrug-resistant strains. And cytotoxic activity assay showed that the compounds tested did not affect cell viability on the Human cervical (HeLa) cells at their MICs. The current study therefore suggests that 5-aryloxypyrazoles bearing a rhodanine-3-aromatic acid moiety are promising scaffolds for the development of novel Gram-positive antibacterial agents.

Full text of DOI:10.1016/j.bmcl.2012.09.107

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7024–7028
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 5-aryloxypyrazole derivatives bearing
a rhodanine-3-aromatic acid as potential antimicrobial agents
Chang-Ji Zheng a, , Ming-Xia Song a, , Liang-Peng Sun a, Yan Wu a, Lan Hong b, 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 133002,
PR China
b Yanbian University College of Medicine, 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:
Received 25 July 2012
Revised 7 September 2012
Accepted 28 September 2012
Available online 6 October 2012
Three novel series of 5-aryloxypyrazole derivatives have been synthesized and tested for their antibacte-
rial activity. The majority of the synthesized compounds showed potent inhibitory activity against Gram-
positive bacteria Staphylococcus aureus 4220, especially against the strains of multidrug-resistant clinical
isolates (MRSA3167/3506 and QRSA3505/3519). Among which compounds IIIb, IIIg and IIIm showed the
most potent levels of activity (MIC = 1 lg/mL) against the multidrug-resistant strains. And cytotoxic
activity assay showed that the compounds tested did not affect cell viability on the Human cervical
(HeLa) cells at their MICs. The current study therefore suggests that 5-aryloxypyrazoles bearing a rhoda-
nine-3-aromatic acid moiety are promising scaffolds for the development of novel Gram-positive antibac-
terial agents.
Keywords:
Rhodanine
5-Aryloxypyrazole
Antibacterial activity
Ó 2012 Elsevier Ltd. All rights reserved.
Although several different classes of antibacterial agents are
currently available, pathogenic bacteria can effectively develop
resistance to these drugs in the majority of cases. To maintain
some level of control over this serious medical problem, the devel-
opment and elaboration of new types of antibacterial agents is a
very important and necessary task.1
Pyrazoles represent a key structural motif in heterocyclic chem-
istry and occupy a significant position in medicinal and pesticide
chemistry because of their capability to exhibit a wide range of
biological activities, including antibacterial,2–5 anti-viral, anti-
tumor,6–8 anti-inflammatory,9 analgesic,10 fungistatic,11 and anti-
hyperglycemic activity.12,13 The use of pyrazole derivatives as
potential antimicrobial agents has received considerable attention
following the discovery of the natural pyrazole C-glycoside, pyra-
zofurin that demonstrated a broad spectrum of antimicrobial activ-
ity.14 Rhodanine and its derivatives have also been reported to
exhibit a broad spectrum of biological activities, behaving as anti-
bacterial,15–17 antifungal,18 antidiabetic,19 antitubercular,20,21 anti-
HIV,22,23 antiparasitic,24 hypnotic,25 and anthelmintic agents.26,27
Previously, we reported the identification of a 1,3-diarylpyraz-
this, we reported the identification of a phenylalanine-derived rho-
danine derivative (compound B, MIC = 1 g/mL) that showed high
l
levels of inhibitory activity against methicillin-resistant Staphylococcus
aureus (MRSA) and quinoline-resistant Staphylococcus aureus
(QRSA).29 Herein, we describe further modifications that we have
made to compounds A and B, using the molecule hybrid approach.
These changes were focused on preserving the rhodanine and
1-phenylpyrazole moieties and substituting the fatty acid moiety
at the N3-position of the rhodanine with different aromatic acids.
Furthermore, we wanted to substitute the phenyl ring at the
3-position of the pyrazole ring with a methyl group and to intro-
duce an aryloxy group at the 5-position of the pyrazole ring
while simultaneously investigating variations to the aryloxy ring
substituents (Fig. 1). Thus, 48 new pyrazole derivatives bearing
aromatic acid-derived rhodanine moieties (I–III) were synthesized,
characterized and evaluated as antimicrobial agents.
The target compounds were synthesized according to the route
depicted in Scheme 1. Briefly, ethyl acetoacetate was reacted with
phenylhydrazine to afford 3-methyl-1-phenyl-1H-pyrazol-5-ol (2).
Compound 2 was treated with phosphorus oxychloride to give 5-
chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (3), which
was subsequently reacted with different substituted phenols to
provide the intermediates 4a–p. Compounds 4a–p were then sub-
jected to a Knoevenagel condensation reaction with the appropri-
ate rhodanine-3-aromatic acids, which had themselves been
synthesized using the reported procedure,30 to provide three new
series of target compounds (I–III). The structures of the desired
compounds were confirmed by IR, 1H NMR, 13C NMR, mass spectral
and elemental analyses.31
ole derivative (compound A, MIC = 2 lg/mL), which was strongly
active against several strains of Gram-positive bacteria (including
multidrug-resistant clinical isolates).28 The study of these types
of compounds and their associated antimicrobial properties has
been central to our ongoing research efforts. Following on from
⇑
Corresponding author. Tel.: +86 433 2435003; fax: +86 433 2435004.
E-mail address: piaohuri@yahoo.com.cn (H.-R. Piao).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.107

C.-J. Zheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7024–7028
7025
Ph
COOH
Ar
COOH
S
COOH
O
O
O
N
S
N
N
S
S
Cl
S
S
H3C
O
N
Cl
N
R
N
N
O
O
F
compound A
target compounds
compound B
Figure 1. Lead compound and structure-based design of the target compound.
CHO
Cl
H3C
H3C
OH
O
O
1. PhNHNH2/C2H5OH
N
POCl3
DMF
N
N
N
O
2. conc.HCl
3.10%NaOH
1
2
3
Ar
Ar
COOH
O
COOH
O
CHO
O
N
H3C
N
S
S
S
S
ROH/K2CO3
DMF
N
H3C
N
O
CH3COOH
C2H5OH, relux
R
N
N
R
4a-p
Ia-p, IIa-p, IIIa-p
Ar: Ia-p:
IIa-p:
HO
IIIa-p:
N
H
e: 4-Cl
f: 4-F
m: 2,4-(CH3)2
n: 3-CF3
i: H
a: 2,6-(Cl)2
b: 2,4-(Cl)2
c: 2-Cl
R:
j: 2-CH3
g: 4-Br
o: 4-OCH3
p: 2-OCH3
k:3-CH3
l: 4-CH3
h: phenyl (3,4-fused)
d: 3-Cl
Scheme 1. Synthetic scheme for the synthesis of the target compounds I–III.
The synthesized compounds were tested for their antimicrobial
activity against selected Gram-positive, Gram-negative, and multi-
drug-resistant bacteria. The results have been presented in Tables
1–4. In a preliminary assay, the intermediates (4a–p) did not exhi-
clinical isolates of multidrug-resistant Gram-positive bacterial
strains. Fortunately, the compounds synthesized exhibited a higher
inhibitory activity against the multidrug-resistant Gram-positive
bacterial strains than the other Gram-positive bacterias (S. aureus
4220, 209, 503) and Gram-negative strains (E. coli 1356). The
resulting data has been reported in Tables 3 and 4. As shown in
Table 3, compounds Ia–p exhibited significant activity against four
bit any antibacterial activity at 64 lg/mL. As shown in Tables 1 and
2,32 all of the synthesized compounds (I–III) were screened for
their activities against three different strains of Gram-positive bac-
teria (S. aureus RN 4220, S. aureus KCTC 209 and S. aureus KCTC 503)
and one Gram-negative strain (Escherichia coli 1356). It was clear
from the results that most of the synthesized compounds potently
MRSA and QRSA strains with MIC values of 1–8
ing activities 2, 4 and eightfold greater than that of norfloxacin.
Compound Ib, in particular, showed MIC values of 1 and 2 g/mL
against MRSA and QRSA, respectively, representing an eightfold in-
crease in potency relative to norfloxacin (MIC = 8 g/mL and 4 g/
mL) and a 32-fold increase relative to oxacillin (MIC >64 g/mL).
Compounds IIa–p showed moderate activity with MIC values of
8–32 g/mL against the four multidrug-resistant Gram-positive
bacterial strains tested. In Table 4, compounds IIIa–p also exhib-
ited significant activity with MIC values of 1–8 g/mL. Compounds
IIIb, IIIg and IIIm were more potent than both standard drugs (oxa-
cillin and norfloxacin) with MIC values of 1 g/mL against MRSA
strains, representing a 4 to eightfold increase in activity relative
to norfloxacin (MIC = 8 g/mL and 4 g/mL) and a 64-fold increase
lg/mL, represent-
l
inhibited S. aureus RN 4220 with MIC values in the range of 2–8 lg/
mL. Of the compounds tested, compounds Ib, Ik, Il, Im, IIIb, IIIe,
IIIg, IIIh and IIIm showed high levels of inhibitory activity against
S. aureus RN 4220 with a MIC value of 2 lg/mL, which was compa-
rable to that of the positive control, norfloxacin. Unfortunately,
none of the compounds tested showed any inhibitory activity
against the Gram-positive strains, S. aureus KCTC 209 and 503,
l
l
l
l
l
and the Gram-negative strain, E. coli 1356 (MICs >64
Compounds that exhibited inhibitory activity against the Gram-
positive bacteria with MIC values of no greater than 32 g/ml were
selected and evaluated for their inhibitory potencies against the
lg/mL).
l
l
l
l

7026
C.-J. Zheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7024–7028
Table 1
Inhibitory activity (MIC,
lg/mL) of compounds Ia–p and IIa–p against bacteria
Compd
R
Gram-positive strains
209
Gram-negative strains
S. aureus
E. coli
4220
503
1356
Ia
Ib
Ic
Id
Ie
If
Ig
Ih
Ii
Ij
Ik
Il
Im
In
Io
Ip
IIa
IIb
IIc
IId
IIe
IIf
IIg
IIh
IIi
IIj
IIk
IIl
IIm
IIn
IIo
IIp
2,6-(Cl)2
2,4-(Cl)2
2-Cl
3-Cl
4-Cl
4
2
8
4
4
8
2
2
8
4
4
4
2
4
8
16
16
8
>32
16
16
>32
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>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
>64
>64
>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
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>16
>64
4-F
4-Br
Phenyl(3,4-fused)
H
2-CH3
3-CH3
4-CH3
2,4-(CH3)2
3-CF3
4-OCH3
2-OCH3
2,6-(Cl)2
2,4-(Cl)2
2-Cl
3-Cl
4-Cl
4-F
4-Br
Phenyl(3,4-fused)
H
8
>32
16
16
16
8
16
>32
>32
2
2-CH3
3-CH3
4-CH3
2,4-(CH3)2
3-CF3
4-OCH3
2-OCH3
Norfloxacin
Oxacillin
—
1
1
1
Table 2
Inhibitory activity (MIC,
l
g/mL) of compounds IIIa–p against bacteria
Compd
R
Gram-positive strains
209
Gram-negative strains
S. aureus
E. coli
4220
503
1356
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
IIIg
IIIh
IIIi
IIIj
IIIk
IIIl
IIIm
IIIn
IIIo
IIIp
Norfloxacin
Oxacillin
2,6-(Cl)2
2,4-(Cl)2
2-Cl
3-Cl
4-Cl
4
2
4
4
2
8
2
2
8
8
4
4
2
4
8
16
2
1
>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
>64
>64
2
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
4-F
4-Br
Phenyl(3,4-fused)
H
2-CH3
3-CH3
4-CH3
2,4-(CH3)2
2-CF3
4-OCH3
2-OCH3
1
1
>64
relative to oxacillin (MIC >64
l
g/mL). For the QRSA strains, com-
was III > I > II, which indicated that the introduction of a hydroxyl
group on the aryl ring (II) did not have a positive impact on the
activity of the compounds. Relative to the previously reported
compounds bearing a rhodanine-3-fatty acid moiety (compound
A), the current 1,3-diphenyl-1H-pyrazole derivatives bearing a
rhodanine-3-aromatic acid moiety (I and III) generally exhibited
pounds IIIb, IIIg and IIIm also presented high levels of activity with
MIC values equal to that of oxacillin (MIC = 1 g/mL) but much
greater than norfloxacin (MIC >64 g/mL).
A comparison of the activities across the three different series of
compounds revealed that the general order of inhibitory activity
l
l

C.-J. Zheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7024–7028
7027
Table 3
Inhibitory activity (MIC,
l
g/mL) of compounds Ia–p and IIa–p against clinical isolates of multidrug-resistant Gram-positive strains
Compd
R
Multidrug-resistant Gram-positive strains
MRSA
QRSA
3167
3506
3505
3519
Ia
Ib
Ic
2,6-(Cl)2
2,4-(Cl)2
2-Cl
4
1
8
8
1
8
8
2
8
8
2
8
Id
3-Cl
4
4
4
8
Ie
4-Cl
2
4
4
4
If
4-F
4
8
8
8
Ig
4-Br
2
2
2
2
Ih
Ii
Phenyl(3,4-fused)
H
2
4
2
4
2
8
2
8
Ij
2-CH3
4
8
8
8
Ik
3-CH3
4
4
4
8
Il
4-CH3
4
8
4
4
Im
In
2,4-(CH3)2
3-CF3
2
4
2
4
2
4
2
8
Io
Ip
IIa
IIb
IId
IIe
IIg
4-OCH3
2-OCH3
2,6-(Cl)2
2,4-(Cl)2
3-Cl
4
8
8
8
8
16
8
4
8
8
8
16
8
8
8
8
8
8
16
8
16
8
16
8
32
16
8
4-Cl
4-Br
8
IIh
IIj
IIk
IIl
Phenyl(3,4-fused)
2-CH3
3-CH3
4-CH3
2,4-(CH3)2
3-CF3
8
8
16
8
8
8
16
4
>64
8
8
16
16
8
8
16
8
32
16
16
8
32
>64
1
32
16
16
8
32
>64
1
IIm
IIn
Norfloxacin
Oxacillin
>64
Table 4
Inhibitory activity (MIC,
lg/mL) of compounds IIIa–p against clinical isolates of multidrug-resistant Gram-positive strains
Compd
R
Multidrug-resistant Gram-positive strains
MRSA
QRSA
3167
3506
3505
3519
IIIa
IIIb
IIIc
2,6-(Cl)2
2,4-(Cl)2
2-Cl
4
1
2
4
1
4
4
1
4
4
1
4
IIId
3-Cl
2
4
4
4
IIIe
4-Cl
2
2
2
2
IIIf
4-F
4
4
8
8
IIIg
4-Br
1
1
1
1
IIIh
IIIi
Phenyl(3,4-fused)
H
2
8
2
8
2
8
2
8
IIIj
IIIk
IIIl
IIIm
IIIn
2-CH3
3-CH3
4-CH3
2,4-(CH3)2
3-CF3
2
4
2
1
4
4
2
1
8
8
4
1
8
8
4
1
4
4
4
8
IIIo
IIIp
4-OCH3
2-OCH3
4
8
4
8
8
8
8
8
Norfloxacin
Oxacillin
8
>64
4
>64
>64
1
>64
1
higher levels of inhibitory activity. The current results therefore
supported the hypothesis of Hardej et al., who suggested that the
presence of a phenylalanine moiety at the N3-position would in-
crease the likelihood of bacterial cell wall penetration and there-
fore potentially improve the anti-MRSA activity of such
compounds.30
In addition, some clear structure activity relationship patterns
were found between the antibacterial activity and the position
and physicochemical properties of the different substituents on
the phenyl ring. The introduction of some halogen groups on the
phenyl ring generally increased the antimicrobial activity, relative
to the non-substituted example. A comparison of the halogen
derivatives at the 4-position of the phenyl ring indicated that the
different halogen atoms contributed to the antimicrobial activity
in the order of Br > Cl > F. The position of the Cl substituent on
the phenyl ring also affected the activity of the compounds with
an activity order of 2,4-(Cl)2 > 4-Cl > 2-Cl = 3-Cl > 2,6-(Cl)2. A com-
parison of the compounds not containing halogens that exhibited
lower levels of activity showed an activity order of 2,4-
(CH3)2 > 4-CH3 > 2-CH3 > 3-CH3 = 4-OCH3 > 2-OCH3 = H.
The cytotoxic properties of compounds Ib and IIIb were also
investigated using MTT colorimetric assay to determine if their

7028
C.-J. Zheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7024–7028
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J.; Leonetti, J. P. J. Antimicrob. Chemother. 2006, 58, 778.
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Nogueras, M.; Rodero, L.; Garibotto, F. M.; Enriz, R. D.; Zacchino, S. A. Bioorg.
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Cytotoxic activity of compounds Ib and IIIb against HeLa cell
a
Compound
IC50 (lg/mL)
Ib
IIIb
3.54
5.76
a
IC50 is the concentrations required to inhibit 50% of cell growth.
20. Murugan, R.; Anbazhagan, S.; Lingeshwaran, S.; Narayanan, S. Eur. J. Med. Chem.
2009, 44, 3272.
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observed antibacterial activity was caused by selective toxicity to-
wards the bacterial cells and the results are shown in Table 5.33
Compounds Ib and IIIb did not affect cell viability on the Human
22. Brooke, E. W.; Davies, S. G.; Mulvaney, A. W.; Okada, M.; Pompeo, F.; Sim, E.;
Vickers, R. J.; Westwood, I. M. Bioorg. Med. Chem. 2003, 13, 2527.
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cervical (HeLa) cells at their MICs (1 or 2 lg/mL) but showed cyto-
toxicity at much higher concentrations. The inconsistency of com-
pounds Ib and IIIb between their antibacterial activity and
cytotoxicity suggests that there may be an antibacterial mecha-
nism different from cytotoxicity.
In summary, we have synthesized three new series of 5-aryloxy
pyrazole derivatives (I–III) and evaluated their antibacterial activ-
ities against Gram-positive and Gram-negative bacterias. The
majority of the synthesized compounds showed potent inhibitory
activity against Gram-positive bacteria S. aureus 4220, especially
against the strains of multidrug-resistant clinical isolates
(MRSA3167/3506 and QRSA3505/3519). Among which compounds
IIIb, IIIg and IIIm showed the most potent levels of activity
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31. Preparation of IIIb: A mixture of 4b (104 mg, 3 mmol), appropriate rhodanine-
3-aromatic acid (96 mg, 3 mmol), 10 drops glacial acetic acid and 10 drops
piperidine in ethanol (20 mL) was refluxed for 4 h. After cooling, the solvent
was evaporated in vacuo, followed by the purification of the resulting residue
by silica gel column chromatography (dichloromethane/methanol, 100:1) to
afford IIIb (344 mg, 53%) as yellow solid. Mp: 185–187 °C. IR (KBr) cmÀ1: 3406
(OH), 3074 (NH), 1712 (C@O). 1H NMR (DMSO-d6, 300 MHz, ppm): d 2.43 (s,
3H, CH3), 3.56–3.73 (m, 2H, CHCH2), 5.55 (br s, 1H, CH2CH), 6.63–7.69 (m, 14H,
Ar-H), 10.68 (s, 1H, NH), 12.51 (s, 1H, COOH). 13C NMR (DMSO-d6, 300 MHz,
ppm): d 193.92, 172.17, 169.46, 166.75, 150.48, 149.95, 143.69, 136.14, 135.94,
130.38, 129.34, 128.75, 128.47, 128.29, 127.25, 122.75, 122.43, 122.23, 120.98,
120.63, 119.82, 118.09, 117.03, 116.34, 111.26, 105.00, 61.25, 21.29, 12.92. MS
(APCI) m/z 649 (M+1). Anal. Calcd. for C31H22Cl2N4O4S2: C, 57.32; H, 3.41; N,
8.63. Found: C, 57.15; H, 3.34; N, 8.76.
32. Anti-bacterial activity assay: The micro-organisms used in the present study
were S. aureus (S. aureus RN 4220, S. aureus KCTC 209, S. aureus KCTC 503), and
Escherichia coli (E. coli 1356). The strains of multidrug-resistant clinical isolates
were methicillin-resistant Staphylococcus aureus (MRSA CCARM 3167 and MRSA
CCARM 3506) and quinolone-resistant Staphylococcus aureus (QRSA CCARM
3505 and QRSA CCARM 3519). Clinical isolates were collected from various
patients hospitalized in several clinics. The in vitro anti-bacterial activity was
evaluated using a 96-well microtiter plate and a serial dilution method to
obtain the Minimum Inhibitory Concentration (MIC) with different strains
including multidrug-resistant clinical isolates. Oxacillin and norfloxacin were
used as positive controls. Test bacteria were grown to mid-log phase in
Mueller-Hinton broth (MHB) and diluted 1000-fold in the same medium. The
bacteria of 105 CFU/mL were inoculated into MHB and dispensed at 0.2 mL/
(MIC = 1 lg/mL) against all of the multidrug-resistant clinical iso-
lates tested. Compounds Ib and IIIb were evaluated for their cyto-
toxicity and exhibited no significant influence on cell viability in
the HeLa cells at their MICs. The mechanism of action of these com-
pounds remains unknown and efforts to establish the cause of their
antibacterial activity are currently underway in our laboratories
and will be reported in due course.
Acknowledgment
This work was supported by the National Science Foundation of
China (Grant number 20962021).
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