Synthesis and antibacterial evaluation of rhodanine-based 5-aryloxy pyrazoles against selected methicillin resistant and quinolone-resistant Staphylococcus aureus (MRSA and QRSA)

Article abstract of DOI:10.1016/j.ejmech.2012.12.007

With an intention to synergize the anti-bacterial activity of 5-aryloxy pyrazole and rhodanine derivatives, eight series of hybrid compounds have been synthesized and evaluated for their antibacterial activity. The majority of the synthesized compounds showed good inhibitory activity against selected methicillin resistant and quinolone-resistant Staphylococcus aureus (MRSA, QRSA) with minimum inhibitory concentration (MIC) values in the range of 1-32 μg/mL. The cytotoxicity test suggests that these compounds exhibited in vitro antibacterial activity at non-cytotoxic concentrations. These studies therefore suggest that rhodanine-based 5-aryloxy pyrazoles are interesting scaffolds for the development of novel Gram-positive antibacterial agents.

Full text of DOI:10.1016/j.ejmech.2012.12.007

European Journal of Medicinal Chemistry 60 (2013) 376e385
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antibacterial evaluation of rhodanine-based 5-aryloxy
pyrazoles against selected methicillin resistant and quinolone-
resistant Staphylococcus aureus (MRSA and QRSA)
,
Ming-Xia Song a b, Chang-Ji Zheng a, Xian-Qing Deng a, Liang-Peng Sun a, Yan Wu a, Lan Hong c,
,
Ying-Jing Li a, Yi Liu a, Zhi-Yu Wei a, Ming-Jun Jin 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,
977 Gongyuan Road, Jilin, Yanji 133002, PR China
b Jinggangshan University College of Medicine, Ji’an 343100, PR China
c 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:
With an intention to synergize the anti-bacterial activity of 5-aryloxy pyrazole and rhodanine deriva-
tives, eight series of hybrid compounds have been synthesized and evaluated for their antibacterial
activity. The majority of the synthesized compounds showed good inhibitory activity against selected
methicillin resistant and quinolone-resistant Staphylococcus aureus (MRSA, QRSA) with minimum
Received 25 July 2012
Received in revised form
24 November 2012
Accepted 5 December 2012
Available online 20 December 2012
inhibitory concentration (MIC) values in the range of 1e32 mg/mL. The cytotoxicity test suggests that
these compounds exhibited in vitro antibacterial activity at non-cytotoxic concentrations. These studies
therefore suggest that rhodanine-based 5-aryloxy pyrazoles are interesting scaffolds for the develop-
ment of novel Gram-positive antibacterial agents.
Keywords:
Rhodanine
5-Aryloxy pyrazole
Anti-bacterial
MRSA
Ó 2012 Elsevier Masson SAS. All rights reserved.
QRSA
1. Introduction
against several strains of Gram-positive bacteria (including
multidrug-resistant clinical isolates) [11]. The study of these types
An alarming increment in pathogenic resistance to existing anti-
microbial agents is a serious problem and necessitates continuing
research into new classes of antimicrobials [1]. This crops up an
enormous interest in antibacterial research and we strongly believe
that there is an urgent call for the development of new antibacterial
drugs with novel structure and a mechanism of action possibly
different from that of current first-line antimicrobial drugs [2].
5-Aryloxy pyrazoles are chemically useful synthons bearing
diverse biological activities, including antimicrobial [2], analgesic
and anti-inflammatory activity [3]. Rhodanine and its derivatives
have also been reported to exhibit a broad spectrum of biological
activities, behaving as antibacterial [4e6], antifungal [7], antipar-
asitic [8], and anthelmintic agents [9,10].
of compounds and their associated antimicrobial properties has
been central to our ongoing research efforts. Herein, we describe
further modifications that we have made to compound A, using the
molecule hybrid approach. These modifications were focused on
preserving the rhodanine and 1-phenylpyrazole moieties and
substituting the acetic acid moiety at the N3-position of the rho-
danine with several different fatty acids. Meanwhile, we introduced
an aryloxy group at the 5-position of the pyrazole ring while
simultaneously investigating variations to the aryloxy ring
substituents. For the convenience of synthesis, we used ethyl ace-
toacetate as a starting material, which was eventually resulted in
a methyl group at the 3-position of the pyrazole ring (Fig. 1). Thus,
eight novel series of rhodanine-based 5-aryloxy pyrazoles (5e12)
were synthesized, characterized and screened for their antibacte-
rial activities. Moreover, in order to check out whether the
dichlorophenyl ring on 3-position of the pyrazole ring is essential
for activity or not, a series of (Z)-2-(5-((5-chloro-3-methyl-1-
phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic acids (13) were also designed.
Previously, we reported the identification of a 1,3-diarylpyrazole
derivative (compound A, MIC ¼ 4
mg/mL), which was strongly active
* Corresponding author. Tel.: þ86 433 2435003; fax: þ86 433 2435004.
E-mail address: piaohuri@yahoo.com.cn (H.-R. Piao).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.007

M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
377
Fig. 1. Lead compound and structure-based design of the target compounds.
2. Chemistry
In the present studies, three series of compounds (5, 6 and 7)
were firstly synthesized and evaluated for their antibacterial
activity, among which compounds 5b, 6b and 7b with 2,4-(Cl)2
substituents, compounds 5g, 6g and 7g with 4-Br substituent, and
compounds 5m, 6m and 7m with 2,4-(CH3)2 substituents presented
much more potent activity in their respective series. Based on this
fact, we inferred that the presence of 2,4-(Cl)2, 2,4-(CH3)2, or 4-Br
moieties may have more effects than the other substituents on the
anti-microbial activity of these compounds. Therefore, in the next
five series (8e12), only several compounds with 2,4-(Cl)2, and/or 4-
Br, and/or 2,4-(CH3)2 group were prepared to avoid a repetition.
Based on the analysis of the activities of the synthesized
compounds, the following structureeactivity relationships (SARs)
were obtained. Introduction of some halogen groups on the phenyl
ring generally increased the antimicrobial activity, relative to the
non-substituted example. In the chlorine-substituted compounds,
2,4-(Cl)2 groups had a good contribution to the activity (like
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 (4). Compounds 3 or 4 were then
subjected to
a Knoevenagel condensation reaction with the
appropriate rhodanine-3-fatty acids, which had themselves been
synthesized using the reported procedure [11], to provide nine
new series of target compounds (5e13). The structures of the
desired compounds were confirmed by IR, 1H NMR, 13C NMR and
mass spectral.
3. Results and discussion
compound 7b with 2,4-(Cl)2 groups, MIC ¼ 2
mg/mL). 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 generally in an order of Br > Cl > F. In the
series 5, for example, the MIC values of compound 5g (4-Br),
compound 5e (4-Cl), and compound 5f (4-F) were 8, 16, and
3.1. Anti-bacterial activity
The antimicrobial assay was carried out using the following
bacterial strains methicillin-resistant Staphylococcus aureus CCARM
3167 and 3506, and quinolone-resistant S. aureus CCARM 3505 and
3519. 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).
The title compounds along with the standard drugs (oxacillin
and norfloxacin) for comparison were evaluated for their inhibitory
activity against selected methicillin-resistant S. aureus (MRSA) and
quinolone-resistant S. aureus (QRSA). The biological results
revealed that no compound in series 9e12 showed any inhibitory
>64 mg/mL, respectively. The similar trend was found in the series 6
and 7. For the derivatives bearing electron-donating substituents, it
seems that the methyl group displays much more impact on the
antibacterial activity than the methoxy group with an 1e4 fold
increase in potency. In the series 6, for another example, the MIC
values of the compound 6l with 4-CH3, compound 6o with 4-OCH3
were 4 and 8 mg/mL, respectively. A comparison of the activities
across the eight different series of compounds, a general inhibitory
activity order of series 7 > series 6, 8 > series 5 > series 9, 10, 11, 12
was obtained, which indicated that the increase of polar groups at
the N3-position of the rhodanine ring did not exhibit a positive
impact on the activity of the compounds. Relative to the previously
reported compound bearing a rhodanine-3-acetic acid moiety
(compound A), the current rhodanine-based 5-aryloxy pyrazoles
generally exhibited higher levels of inhibitory activity [12].
Compared the activity of 13aed with that of the compound A and
compounds 5e8, it can be found that a substituted phenyl ring on
pyrazole ring is essential for activity, but its position is not fixed.
The substituted phenyl rings on both side of the pyrazole ring (3 or
5 position) afforded a good contribution to the activity.
activity at 64 mg/mL against the selected drug-resistant organisms.
For this reason, Tables 1 and 2 only presented the results of
compounds of the series 5e8. As shown in Tables 1 and 2,
compounds in series 5 exhibited moderate activity, most of which
with MICs of 8, 16 or 32
better activity against MRSA CCARM 3167 with a MIC value of 4
mL. Compounds in series 6 and 8 presented the high activity with
MIC values of 2e8 g/mL, making them slightly less active than
g/mL) generally. Most
mg/mL. Only compound 5b displayed much
mg/
m
compounds in series 7 (MIC ¼ 1e4
m
compounds in series 6, 7 and 8 presented comparable or much
more potent activities than norfloxacin. Compound 7b, in partic-
ular, showed MICs of 1
increase in potency relative to norfloxacin (MIC ¼ 8
mL) and a 64-fold increase relative to oxacillin (MIC > 64
For the QRSA strains, compound 7b also presented high levels of
with MIC values slightly less active than oxacillin (MIC ¼ 1 g/mL)
but much greater than norfloxacin (MIC > 64 g/mL).
m
g/mL against MRSA, representing an 8-fold
g/mL and 4 g/
g/mL).
m
m
m
3.2. Cytotoxicity
m
To see whether the antibacterial activity of compounds 6b, 7b
and 8b is selectively toxic to bacteria, their cytotoxicity was
m

378
M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
Scheme 1. Synthetic scheme for the synthesis of the target compounds 5e13.
evaluated (Table 3). Compounds 6b, 7b and 8b did not affect cell
viability on the Human cervical (HeLa) cells at their MICs (4, 1 or
levels of activity (MIC ¼ 1 or 2
mg/mL) against selected MRSA and
QRSA strains. While the mechanism of action of this compound
remains unknown, efforts to establish the cause of their antibac-
terial activity are ongoing and will be reported in due course.
2
mg/mL, respectively) but showed cytotoxicity at much higher
concentrations. The inconsistency of compounds 6b, 7b and 8b
between their antibacterial activity and cytotoxicity suggests that
compounds 6b, 7b and 8b exhibited in vitro antibacterial activities
at non-cytotoxic concentrations.
5. Experimental protocols
5.1. Chemistry
4. Conclusion
Melting points were determined in open capillary tubes and are
uncorrected. Reaction courses were monitored by TLC on silica gel-
precoated F254 Merck plates. Developed plates were examined with
UV lamps (254 nm). IR spectra were recorded (in KBr) on a FTIR1730.
1H NMR spectra were measured on a Bruker AV-300 spectrometer
using TMS as the internal standard. Mass spectra were measured on
Based on our previous work, we have synthesized eight new
series of rhodanine derivatives (5e12) and evaluated them for
antibacterial and cytotoxic activities. Most of the compounds
showed good antibacterial activities against multidrug-resistant
strains of clinical isolates. Compound 7b showed the most potent

M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
379
Table 1
5.3. General procedure for the preparation of compound 3
Inhibitory activity (MIC, mg/mL) of compounds 5 and 6 against clinical isolates of
multidrug-resistant Gram-positive strains.
To a cold, stirred solution of dimethylformamide (1 mmol) and
phosphorous oxychloride (6 mmol) was added compound 2
(1 mmol). The reaction mixture was stirred at 80 ꢀC for 3 h, cooled
to room temperature, poured into ice cold water whereupon a solid
separated out that was filtered, washed with excess of cold water,
dried to afford a yellow solid 5-chloro-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde (3).
5.4. General procedure for the preparation of compounds 4
To a solution of compound 3 (10 mmol) and K2CO3 (15 mmol) in
dry dimethylformamide (10 mL), corresponding substituted
phenols (11 mmol) was added and mixture was stirred overnight at
80 ꢀC. After the completion of reaction, poured the mixture into
100 mL ice-water and filtered to obtain a white solid. The resulting
crude solid was directly used in the next step without purification.
Compound
R0
Multidrug-resistant Gram-positive strains
MRSA
QRSA
3167
3506
3505
3519
5a
5b
5c
5d
5e
5f
2,6-(Cl)2
2,4-(Cl)2
2-Cl
3-Cl
4-Cl
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
2-CH3
3-CH3
4-CH3
2,4-(CH3)2
3-CF3
4-OCH3
2-OCH3
e
32
4
32
8
32
8
32
8
16
16
16
>64
8
16
16
16
>64
8
32
8
16
>64
16
8
16
16
16
>64
8
5.5. General procedure for the preparation of compounds 5
A mixture of 4 (3 mmol), 2-(4-oxo-2-thioxothiazolidin-3-yl)
acetic acid (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 (dichloro-
methane/methanol, 100:1) to get a yellow solid.
5g
5h
8
8
8
5i
5j
5k
5l
>64
16
16
32
8
16
>64
>64
8
>64
32
16
32
8
32
>64
>64
16
4
>64
32
32
32
16
32
>64
>64
8
>64
32
16
32
16
32
>64
>64
16
4
5m
5n
5o
5p
6a
6b
6c
6d
6e
6f
5.5.1. (Z)-2-(5-((5-(2,6-Dichlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5a)
Yield 59%; m.p. 214e216 ꢀC. IR (KBr) cmꢁ1: 3417 (OH), 1705 (C]
4
4
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.35 (s, 3H, CH3), 4.68 (s,
4
4
8
4
4
4
8
4
2H, NCH2), 7.28e7.49 (m, 9H, AreH and CH), 13.41 (s, 1H, COOH).
MS m/z 520 (M þ 1).
4
4
4
4
8
8
8
8
6g
6h
2
4
4
4
4
8
8
8
5.5.2. (Z)-2-(5-((5-(2,4-Dichlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5b)
6i
6j
6k
6l
6m
6n
6o
6p
16
4
16
8
16
8
16
8
Yield 53%; m.p. 196e198 ꢀC. IR (KBr) cmꢁ1: 3426 (OH), 1717 (C]
4
8
4
4
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.46 (s, 3H, CH3), 4.67 (s,
4
4
4
4
2
4
4
2
2H, NCH2), 6.70e7.73 (m, 9H, AreH and CH), 13.40 (s, 1H, COOH).
MS m/z 520 (M þ 1).
4
8
8
8
8
8
8
8
16
8
>64
16
4
>64
16
>64
1
16
>64
1
5.5.3. (Z)-2-(5-((5-(2-Chlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5c)
Norfloxacin
Oxacillin
e
Yield 44%; m.p. 226e228 ꢀC. IR (KBr) cmꢁ1: 3421 (OH), 1718 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.47 (s, 3H, CH3), 4.66 (s,
an HP1100LC (Agilent Technologies, Santa Clara, CA, USA). The major
chemicals were purchased from SigmaeAldrich (St. Louis, MO, USA)
and Fluka Companies (Milwaukee, MI, USA).
2H, NCH2), 6.65e7.58 (m, 10H, AreH and CH), 13.40 (s, 1H, COOH).
MS m/z 486 (M þ 1).
5.5.4. (Z)-2-(5-((5-(3-Chlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5d)
5.2. General procedure for the preparation of compound 2
To a stirred solution of acetylacetic ether (0.1 mol) and 70%
ethanol (5.0 mL), the solution of phenylhydrazine (0.1 mol) with
absolute alcohol (3.0 mL) was added dropwise at 45 ꢀC, and the
mixture was stirred for 20 min at this temperature. Cooled to 20 ꢀC,
added concentrated hydrochloric acid (1.0 mL) dropwise and
reacted for 2 h at 45 ꢀC. Then adjusted the PH value of mixture to 7
using 10% NaOH solution, added 20 mL water and stirred for 1 h at
the room temperature. The precipitated solid was filtered, washed
with cold absolute alcohol to get a white solid 3-methyl-1-phenyl-
1H-pyrazol-5-ol (2).
Yield 44%; m.p. 207e209 ꢀC. IR (KBr) cmꢁ1: 3417 (OH), 1712 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.46 (s, 3H, CH3), 4.67 (s,
2H, NCH2), 6.84e7.62 (m, 10H, AreH and CH), 13.42 (s, 1H, COOH).
MS m/z 486 (M þ 1).
5.5.5. (Z)-2-(5-((5-(4-Chlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5e)
Yield 44%; m.p. 230e232 ꢀC. IR (KBr) cmꢁ1: 3420 (OH),1709 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.46 (s, 3H, CH3), 4.67 (s,

380
M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
Table 2
Inhibitory activity (MIC,
m
g/mL) of compounds 7, 8 and 13 against clinical isolates of multidrug-resistant Gram-positive strains.
Compound
R0
Multidrug-resistant Gram-positive strains
MRSA
QRSA
3167
3506
3505
3519
7a
7b
2,6-(Cl)2
2,4-(Cl)2
8
1
16
1
16
2
16
2
7c
2-Cl
2
4
4
4
7d
3-Cl
4
8
8
8
7e
4-Cl
2
4
4
4
7f
4-F
2
4
8
8
7g
4-Br
2
2
2
2
7h
Phenyl(3,4-fused)
2
2
2
2
7i
H
4
4
8
4
7j
7k
7l
7m
7n
7o
7p
8a
2-CH3
3-CH3
4-CH3
2,4-(CH3)2
3-CF3
4-OCH3
2-OCH3
2,4-(Cl)2
4-Br
2
2
2
2
4
4
16
4
4
4
4
4
2
8
4
16
4
4
4
4
4
2
8
4
16
4
4
4
2
4
2
8
4
16
4
4
8b
8c
13a
2,4-(CH3)2
e
4
4
4
4
>64
16
16
16
8
>64
32
32
32
4
>64
32
16
32
>64
1
>64
32
32
32
>64
1
13b
13c
13d
Norfloxacin
Oxacillin
e
e
e
e
e
>64
>64
2H, NCH2), 6.92e7.62 (m, 10H, AreH and CH), 13.40 (s, 1H, COOH).
13C NMR (DMSO-d6, 300 MHz, ppm):
193.59, 167.32, 166.18,
154.53, 150.61, 144.94, 136.30, 130.14, 129.40, 128.10, 123.34, 122.48,
2H, NCH2), 6.85e7.60 (m, 10H, AreH and CH), 13.42 (s, 1H, COOH).
MS m/z 531 (M þ 1).
d
119.15, 117.07, 110.38, 104.80, 45.06, 12.91. MS m/z 486 (M þ 1).
5.5.8. (Z)-2-(5-((3-Methyl-5-(naphthalen-2-yloxy)-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5h)
5.5.6. (Z)-2-(5-((5-(4-Fluorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5f)
Yield 35%; m.p. 135e137 ꢀC. IR (KBr) cmꢁ1: 3429 (OH), 1720 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.47 (s, 3H, CH3), 4.60 (s,
Yield 45%; m.p. 208e210 ꢀC. IR (KBr) cmꢁ1: 3427 (OH), 1713 (C]
2H, NCH2), 6.52e8.53 (m, 13H, AreH and CH), 13.37 (s, 1H, COOH).
MS m/z 502 (M þ 1).
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.45 (s, 3H, CH3), 4.67 (s,
2H, NCH2), 6.91e7.61 (m, 10H, AreH and CH), 13.41 (s, 1H, COOH).
MS m/z 470 (M þ 1).
5.5.9. (Z)-2-(5-((3-Methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl)
methylene)-4-oxo-2-thioxotetrahydrothiophen-3-yl)acetic acid (5i)
Yield 53%; m.p. 200e202 ꢀC. IR (KBr) cmꢁ1: 3402 (OH),1709 (C]
5.5.7. (Z)-2-(5-((5-(4-Bromophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5g)
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.45 (s, 3H, CH3), 4.65 (s,
2H, NCH2), 6.85e7.61 (m, 11H, AreH and CH), 13.38 (s, 1H, COOH).
MS m/z 452 (M þ 1).
Yield 38%; m.p. 172e174 ꢀC. IR (KBr) cmꢁ1: 3414 (OH), 1712 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.44 (s, 3H, CH3), 4.65 (s,
Table 3
5.5.10. (Z)-2-(5-((3-Methyl-1-phenyl-5-(o-tolyloxy)-1H-pyrazol-4-
yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5j)
Yield 41%; m.p. 191e193 ꢀC. IR (KBr) cmꢁ1: 3407 (OH),
Cytotoxic activity of compounds 6b, 7b and 8b against HeLa cell.
Compound
IC50 (m
g/mL)a
1703 (C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.41 (s,
6b
7b
8b
10.39
6.43
12.33
3H, ph-CH3), 2.46 (s, 3H, CH3), 4.65 (s, 2H, NCH2), 6.37e7.55
(m, 10H, AreH and CH), 13.41 (s, 1H, COOH). MS m/z 466
(M þ 1).
a
IC50 is the concentrations required to inhibit 50% of cell growth.

M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
381
5.5.11. (Z)-2-(5-((3-Methyl-1-phenyl-5-(m-tolyloxy)-1H-pyrazol-
5.6.2. (S,Z)-2-(5-((5-(2,4-Dichlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6b)
4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5k)
Yield 39%; m.p. 158e160 ꢀC. IR (KBr) cmꢁ1: 3408 (OH), 1702 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
2.16 (s, 3H, ph-CH3), 2.45
Yield 43%; m.p. 122e124 ꢀC. IR (KBr) cmꢁ1: 3437 (OH), 1709 (C]
(s, 3H, CH3), 4.66 (s, 2H, NCH2), 6.75e7.62 (m, 10H, AreH and CH),
13.41 (s, 1H, COOH). MS m/z 466 (M þ 1).
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.61 (d, 3H, J ¼ 6.8 Hz,
CHCH3), 1.21 (d, 3H, J ¼ 6.6 Hz, CHCH3), 2.44 (s, 3H, CH3), 2.61e2.72
(m, 1H, CH(CH3)2), 4.80 (s, 1H, NCH), 7.19e7.72 (m, 9H, AreH, and
CH), 11.03 (s, 1H, COOH). MS m/z 563 (M þ 1).
5.5.12. (Z)-2-(5-((3-Methyl-1-phenyl-5-(p-tolyloxy)-1H-pyrazol-4-
yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5l)
Yield 49%; m.p. 207e209 ꢀC. IR (KBr) cmꢁ1: 3426 (OH), 1717 (C]
5.6.3. (S,Z)-2-(5-((5-(2-Chlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6c)
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.16 (s, 3H, ph-CH3), 2.45
(s, 3H, CH3), 4.66 (s, 2H, NCH2), 6.24e7.96 (m, 10H, AreH and CH),
13.40 (s, 1H, COOH). MS m/z 466 (M þ 1).
Yield 54%; m.p. 91e93 ꢀC. IR (KBr) cmꢁ1: 3427 (OH),1706 (C]O).
1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.64 (d, 3H, J ¼ 6.8 Hz,
5.5.13. (Z)-2-(5-((5-(2,4-Dimethylphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic
acid (5m)
CHCH3), 1.21 (d, 3H, J ¼ 6.4 Hz, CHCH3), 2.46 (s, 3H, CH3), 2.60e2.74
(m, 1H, CH(CH3)2), 5.09 (s, 1H, NCH), 6.66e7.57 (m, 10H, AreH, and
CH), 12.66 (s, 1H, COOH). MS m/z 529 (M þ 1).
Yield 43%; m.p. 230e232 ꢀC. IR (KBr) cmꢁ1: 3421 (OH), 1712
(C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
2.12 (s, 3H, ph-CH3),
5.6.4. (S,Z)-2-(5-((5-(3-Chlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6d)
2.37 (s, 3H, ph-CH3), 2.45 (s, 3H, CH3), 4.65 (s, 2H, NCH2), 5.76e
7.95 (m, 9H, AreH and CH), 13.39 (s, 1H, COOH). MS m/z 480
(M þ 1).
Yield 49%; m.p. 94e96 ꢀC. IR (KBr) cmꢁ1: 3443 (OH), 1712 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.64 (d, 3H, J ¼ 6.7 Hz,
5.5.14. (Z)-2-(5-((3-Methyl-1-phenyl-5-(3-(trifluoromethyl)
phenoxy)-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-
3-yl)acetic acid (5n)
CHCH3), 1.16 (d, 3H, J ¼ 6.3 Hz, CHCH3), 2.44 (s, 3H, CH3), 2.61e2.74
(m, 1H, CH(CH3)2), 5.07 (s, 1H, NCH), 6.85e7.61 (m, 10H, AreH, and
CH), 12.63 (s, 1H, COOH). MS m/z 529 (M þ 1).
Yield 40%; m.p. 182e184 ꢀC. IR (KBr) cmꢁ1: 3417 (OH), 1723 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
2.47 (s, 3H, CH3), 4.66 (s,
5.6.5. (S,Z)-2-(5-((5-(4-Chlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6e)
2H, NCH2), 7.18e7.61 (m, 10H, AreH and CH), 13.41 (s, 1H, COOH).
MS m/z 520 (M þ 1).
Yield 46%; m.p. 102e104 ꢀC. IR (KBr) cmꢁ1: 3407 (OH), 1711 (C]
5.5.15. (Z)-2-(5-((5-(4-Methoxyphenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5o)
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.62 (d, 3H, J ¼ 6.8 Hz,
CHCH3), 1.17 (d, 3H, J ¼ 6.3 Hz, CHCH3), 2.44 (s, 3H, CH3), 2.60e2.72
(m, 1H, CH(CH3)2), 4.90 (s, 1H, NCH), 6.91e7.60 (m, 10H, AreH, and
CH), 12.77 (s, 1H, COOH). MS m/z 529 (M þ 1).
Yield 38%; m.p. 188e190 ꢀC. IR (KBr) cmꢁ1: 3413 (OH), 1716 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.45 (s, 3H, CH3), 3.64 (s,
3H, OCH3), 4.67 (s, 2H, NCH2), 6.76e7.95 (m, 10H, AreH and CH),
13.42 (s, 1H, COOH). MS m/z 482 (M þ 1).
5.6.6. (S,Z)-2-(5-((5-(4-Fluorophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6f)
5.5.16. (Z)-2-(5-((5-(2-Methoxyphenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
(5p)
Yield 42%; m.p. 89e91 ꢀC. IR (KBr) cmꢁ1: 3417 (OH), 1712 (C]O).
1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.62 (d, 3H, J ¼ 6.7 Hz,
CHCH3), 1.18 (d, 3H, J ¼ 6.4 Hz, CHCH3), 2.43 (s, 3H, CH3), 2.63e2.72
(m, 1H, CH(CH3)2), 4.87 (s, 1H, CH), 6.92e7.60 (m, 10H, AreH and
CH), 12.67 (s, 1H, COOH). MS m/z 512 (M þ 1).
Yield 38%; m.p. 178e180 ꢀC. IR (KBr) cmꢁ1: 3425 (OH), 1719 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.44 (s, 3H, CH3), 3.84 (s,
3H, OCH3), 4.66 (s, 2H, NCH2), 6.58e7.58 (m, 10H, AreH and CH),
13.42 (s, 1H, COOH). MS m/z 482 (M þ 1).
5.6.7. (S,Z)-2-(5-((5-(4-Bromophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6g)
5.6. General procedure for the preparation of compound 6
Yield 46%; m.p. 120e122 ꢀC. IR (KBr) cmꢁ1: 3414 (OH), 1717 (C]
A
mixture of
4
(3 mmol), (S)-3-methyl-2-(4-oxo-2-
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
CHCH3), 1.16 (d, 3H, J ¼ 6.3 Hz, CHCH3), 2.44 (s, 3H, CH3), 2.60e2.70
(m, 1H, CH(CH3)2), 5.01 (s, 1H, CH), 6.86e7.60 (m, 10H, AreH and
CH), 12.70 (s, 1H, COOH). 13C NMR (DMSO-d6, 300 MHz, ppm):
d
0.63 (d, 3H, J ¼ 6.6 Hz,
thioxothiazolidin-3-yl)butanoic acid (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 get
a yellow solid.
d
193.98, 172.07, 168.83, 166.48, 154.95, 150.58, 144.93, 136.33,
132.97, 129.38, 128.10, 123.17, 122.47, 117.58, 116.05, 104.70, 62.95,
21.97, 21.11, 19.08, 12.90. MS m/z 573 (M þ 1).
5.6.1. (S,Z)-2-(5-((5-(2,6-Dichlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6a)
5.6.8. (S,Z)-3-Methyl-2-(5-((3-methyl-5-(naphthalen-1-yloxy)-1-
phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)butanoic acid (6h)
Yield 53%; m.p. 118e120 ꢀC. IR (KBr) cmꢁ1: 3431 (OH), 1713 (C]
Yield 36%; m.p. 91e93 ꢀC. IR (KBr) cmꢁ1: 3397 (OH),1708 (C]O).
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.63 (d, 3H, J ¼ 6.7 Hz,
1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.55 (d, 3H, J ¼ 6.8 Hz,
CHCH3), 1.22 (d, 3H, J ¼ 6.9 Hz, CHCH3), 2.51 (s, 3H, CH3), 2.61e2.78
(m, 1H, CH(CH3)2), 4.79 (s, 1H, NCH), 7.01e7.53 (m, 9H, AreH, and
CH), 10.73 (s, 1H, COOH). MS m/z 563 (M þ 1).
CHCH3), 1.16 (d, 3H, J ¼ 6.4 Hz, CHCH3), 2.48 (s, 3H, CH3), 2.54e2.65
(m, 1H, CH(CH3)2), 4.74 (s, 1H, CH), 6.51e8.51 (m, 13H, AreH and
CH), 12.35 (s, 1H, COOH). MS m/z 544 (M þ 1).

382
M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
5.6.9. (S,Z)-3-Methyl-2-(5-((3-methyl-5-phenoxy-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)butanoic
acid (6i)
(m,1H, CH(CH3)2), 3.62 (s, 3H, OCH3), 5.00 (s, 1H, CH), 6.80e7.61 (m,
10H, AreH and CH), 13.67 (s, 1H, COOH). MS m/z 524 (M þ 1).
Yield 50%; m.p. 133e135 ꢀC. IR (KBr) cmꢁ1: 3421 (OH), 1702 (C]
5.6.16. (S,Z)-2-(5-((5-(2-Methoxyphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6p)
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.60 (d, 3H, J ¼ 6.6 Hz,
CHCH3), 1.20 (d, 3H, J ¼ 6.2 Hz, CHCH3), 2.43 (s, 3H, CH3), 2.60e2.71
(m, 1H, CH(CH3)2), 4.80 (s, 1H, CH), 6.85e8.15 (m, 11H, AreH and
CH), 11.41 (s, 1H, COOH). MS m/z 494 (M þ 1).
Yield 41%; m.p. 122e124 ꢀC. IR (KBr) cmꢁ1: 3411 (OH), 1716 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.65 (d, 3H, J ¼ 6.5 Hz,
CHCH3), 1.16 (d, 3H, J ¼ 6.3 Hz, CHCH3), 2.42 (s, 3H, CH3), 2.63e2.74
(m,1H, CH(CH3)2), 3.83 (s, 3H, OCH3), 5.05 (s,1H, CH), 6.60e7.56 (m,
10H, AreH and CH), 13.27 (s, 1H, COOH). MS m/z 524 (M þ 1).
5.6.10. (S,Z)-3-Methyl-2-(5-((3-methyl-1-phenyl-5-(o-tolyloxy)-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)
butanoic acid (6j)
Yield 56%; m.p. 96e98 ꢀC. IR (KBr) cmꢁ1: 3399 (OH), 1715 (C]
5.7. General procedure for the preparation of compounds 7
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.62 (d, 3H, J ¼ 6.7 Hz,
CHCH3), 1.16 (d, 3H, J ¼ 6.4 Hz, CHCH3), 2.40 (s, 3H, ph-CH3), 2.45 (s,
3H, CH3), 2.58e2.71 (m, 1H, CH(CH3)2), 4.99 (s, 1H, NCH), 6.37e7.54
(m, 10H, AreH, and CH), 12.27 (s, 1H, COOH). MS m/z 508 (M þ 1).
A
mixture of
4
(3 mmol), (2S)-3-methyl-2-(4-oxo-2-
thioxothiazolidin-3-yl)pentanoic acid (3 mmol), 10 drops glacial
aceticacidand10dropspiperidineinethanol(20mL)wasrefluxedfor
4h.Aftercooling,thesolventwasevaporatedinvacuo,followedbythe
purification of the resulting residue by silica gel column chromatog-
raphy (dichloromethane/methanol, 80:1) to get a yellow solid.
5.6.11. (S,Z)-3-Methyl-2-(5-((3-methyl-1-phenyl-5-(m-tolyloxy)-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)
butanoic acid (6k)
Yield 53%; m.p. 132e134 ꢀC. IR (KBr) cmꢁ1: 3407 (OH), 1719 (C]
5.7.1. (2S)-2-((Z)-5-((5-(2,6-Dichlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylpentanoic acid (7a)
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.63 (d, 3H, J ¼ 6.7 Hz,
CHCH3), 1.16 (d, 3H, J ¼ 6.4 Hz, CHCH3), 2.15 (s, 3H, ph-CH3), 2.44 (s,
3H, CH3), 2.59e2.71 (m, 1H, CH(CH3)2), 5.02 (s, 1H, NCH), 6.27e7.61
(m, 10H, AreH, and CH), 12.80 (s, 1H, COOH). MS m/z 508 (M þ 1).
Yield53%;m.p.106e108 ꢀC. IR(KBr)cmꢁ1:3417(OH),1743(C]O).
1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.63 (t, 3H, J ¼ 7.4 Hz, CH2CH3),
0.80e0.89 (m, 2H, CH3CH2),1.22 (d, 3H, J ¼ 6.4 Hz, CHCH3), 2.15e2.24
(m, 1H, CH3CH), 2.29 (s, 1H, CH3), 4.73 (br.s, 1H, NCH), 7.00e7.53 (m,
9H, AreH and CH), 12.72 (s, 1H, COOH). MS m/z 577 (M þ 1).
5.6.12. (S,Z)-3-Methyl-2-(5-((3-methyl-1-phenyl-5-(p-tolyloxy)-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)
butanoic acid (6l)
5.7.2. (2S)-2-((Z)-5-((5-(2,4-Dichlorophenoxy)-3-methyl-1-
phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)-3-methylpentanoic acid (7b)
Yield 48%; m.p. 140e142 ꢀC. IR (KBr) cmꢁ1: 3405 (OH), 1709 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.64 (d, 3H, J ¼ 6.8 Hz,
CHCH3), 1.14 (d, 3H, J ¼ 6.4 Hz, CHCH3), 2.14 (s, 3H, ph-CH3), 2.43 (s,
3H, CH3), 2.62e2.70 (m, 1H, CH(CH3)2), 5.08 (s, 1H, CH), 6.74e7.60
(m, 10H, AreH and CH), 12.93 (s, 1H, COOH). MS m/z 508 (M þ 1).
Yield 43%;m.p.126e128 ꢀC. IR(KBr)cmꢁ1:3418 (OH),1745(C]O).
1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.74 (t, 3H, J ¼ 7.1 Hz, CH2CH3),
0.81e0.88 (m, 2H, CH3CH2),1.11 (d, 3H, J ¼ 6.4 Hz, CHCH3), 2.37e2.44
(m, 1H, CH3CH), 2.45 (s, 1H, CH3), 5.16 (br.s, 1H, NCH), 6.72e7.73 (m,
9H, AreH and CH), 13.18 (s, 1H, COOH). MS m/z 577 (M þ 1).
5.6.13. (S,Z)-2-(5-((5-(2,4-Dimethylphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6m)
5.7.3. (2S)-2-((Z)-5-((5-(2-Chlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylpentanoic acid (7c)
Yield 44%; m.p. 124e126 ꢀC. IR (KBr) cmꢁ1: 3395 (OH), 1701 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.63 (d, 3H, J ¼ 6.5 Hz,
Yield 54%; m.p. 136e138 ꢀC. IR (KBr) cmꢁ1: 3422 (OH), 1748 (C]
CHCH3), 1.15 (d, 3H, J ¼ 6.2 Hz, CHCH3), 2.10 (s, 3H, ph-CH3), 2.35 (s,
3H, ph-CH3), 2.44 (s, 3H, CH3), 2.63e2.73 (m, 1H, CH(CH3)2), 5.03 (s,
1H, CH), 6.74e7.60 (m, 9H, AreH and CH), 13.27 (s, 1H, COOH). MS
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.74 (t, 3H, J ¼ 7.1 Hz,
CH2CH3), 0.83e0.94 (m, 2H, CH3CH2),1.11 (d, 3H, J ¼ 6.4 Hz, CHCH3),
2.38e2.45 (m, 1H, CH3CH), 2.46 (s, 1H, CH3), 5.15 (d, 1H, J ¼ 8.6 Hz,
NCH), 6.67e7.56 (m, 10H, AreH and CH), 13.15 (s, 1H, COOH). MS m/
z 543 (M þ 1).
m/z 522 (M þ 1). 13C NMR (DMSO-d6, 300 MHz, ppm):
d 194.87,
167.51, 152.14, 150.57, 145.72, 136.50, 132.81, 132.27, 129.23, 128.08,
127.66, 125.14, 123.59, 122.54, 112.48, 109.51, 104.92, 62.87, 44.28,
27.79, 21.97, 19.95, 19.06, 15.79, 12.88.
5.7.4. (2S)-2-((Z)-5-((5-(3-Chlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylpentanoic acid (7d)
5.6.14. (S,Z)-3-Methyl-2-(5-((3-methyl-1-phenyl-5-(3-
(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)butanoic acid (6n)
Yield 49%; m.p. 127e129 ꢀC. IR (KBr) cmꢁ1: 3425 (OH), 1749 (C]
Yield 41%; m.p. 128e130 ꢀC. IR (KBr) cmꢁ1: 3410 (OH), 1706 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.74 (t, 3H, J ¼ 7.4 Hz,
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.61 (d, 3H, J ¼ 6.7 Hz,
CH2CH3), 0.81e0.92 (m, 2H, CH3CH2), 1.17 (d, 3H, J ¼ 6.3 Hz, CHCH3),
2.34e2.42 (m, 1H, CH3CH), 2.43 (s, 1H, CH3), 4.84 (br.s, 1H, NCH),
6.83e7.61 (m, 10H, AreH and CH), 12.55 (s, 1H, COOH). MS m/z 543
(M þ 1).
CHCH3), 1.15 (d, 3H, J ¼ 6.3 Hz, CHCH3), 2.45 (s, 3H, CH3), 2.62e2.70
(m, 1H, CH(CH3)2), 5.03 (s, 1H, CH), 7.18e7.60 (m, 10H, AreH and
CH), 13.45 (s, 1H, COOH). MS m/z 562 (M þ 1).
5.6.15. (S,Z)-2-(5-((5-(4-Methoxyphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylbutanoic acid (6o)
5.7.5. (2S)-2-((Z)-5-((5-(4-Chlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylpentanoic acid (7e)
Yield 41%; m.p. 129e121 ꢀC. IR (KBr) cmꢁ1: 3394 (OH), 1703 (C]
Yield 46%; m.p. 186e188 ꢀC. IR (KBr) cmꢁ1: 3420 (OH), 1741 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.63 (d, 3H, J ¼ 6.6 Hz,
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.72 (t, 3H, J ¼ 7.8 Hz,
CHCH3), 1.16 (d, 3H, J ¼ 6.3 Hz, CHCH3), 2.43 (s, 3H, CH3), 2.61e2.71
CH2CH3), 0.84e1.02 (m, 2H, CH3CH2), 1.17 (d, 3H, J ¼ 6.3 Hz, CHCH3),

M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
383
2.36e2.42 (m, 1H, CH3CH), 2.43 (s, 1H, CH3), 4.81 (br.s, 1H, NCH),
5.7.12. (2S)-3-Methyl-2-((Z)-5-((3-methyl-1-phenyl-5-(p-
6.90e7.60 (m, 10H, AreH and CH), 12.59 (s, 1H, COOH). MS m/z 543
(M þ 1).
tolyloxy)-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)pentanoic acid (7l)
Yield 48%; m.p. 189e191 ꢀC. IR (KBr) cmꢁ1: 3418 (OH), 1755
5.7.6. (2S)-2-((Z)-5-((5-(4-Fluorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylpentanoic acid (7f)
(C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 0.73 (t, 3H,
J ¼ 7.6 Hz, CH2CH3), 0.84e1.03 (m, 2H, CH3CH2), 1.10 (d, 3H,
J ¼ 6.3 Hz, CHCH3), 2.14 (s, 1H, ph-CH3), 2.38e2.42 (m, 1H,
CH3CH), 2.43 (s, 1H, CH3), 5.14 (d, 1H, J ¼ 8.7 Hz, NCH), 6.73e7.59
(m, 10H, AreH and CH), 13.07 (s, 1H, COOH). 13C NMR (DMSO-d6,
Yield 42%; m.p. 164e166 ꢀC. IR (KBr) cmꢁ1: 3413 (OH), 1738 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.74 (t, 3H, J ¼ 7.6 Hz,
CH2CH3), 0.82e0.89 (m, 2H, CH3CH2),1.11 (d, 3H, J ¼ 6.3 Hz, CHCH3),
2.38e2.43 (m, 1H, CH3CH), 2.44 (s, 1H, CH3), 5.16 (d, 1H, J ¼ 7.8 Hz,
NCH), 6.91e7.60 (m,10H, AreH and CH),13.17 (s,1H, COOH). MS m/z
526 (M þ 1).
300 MHz, ppm): d 194.04, 168.67, 166.54, 153.82, 150.65, 145.73,
136.47, 133.25, 130.55, 129.32, 127.97, 124.03, 122.34, 117.41,
114.99, 104.79, 61.54, 32.97, 24.81, 20.01, 17.58, 12.89, 10.84. MS
m/z 522 (M þ 1).
5.7.7. (2S)-2-((Z)-5-((5-(4-Bromophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylpentanoic acid (7g)
5.7.13. (2S)-3-Methyl-2-((Z)-5-((3-methyl-5-phenoxy-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)
pentanoic acid (7m)
Yield 46%; m.p. 188e190 ꢀC. IR (KBr) cmꢁ1: 3426 (OH), 1749 (C]
Yield 50%; m.p. 183e185 ꢀC. IR (KBr) cmꢁ1: 3421 (OH), 1751 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.75 (t, 3H, J ¼ 7.9 Hz,
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.73 (t, 3H, J ¼ 7.8 Hz,
CH2CH3), 0.86e1.02 (m, 2H, CH3CH2), 1.11 (d, 3H, J ¼ 6.4 Hz, CHCH3),
2.37e2.43 (m, 1H, CH3CH), 2.44 (s, 1H, CH3), 5.15 (d, 1H, J ¼ 7.4 Hz,
NCH), 6.86e7.60 (m, 10H, AreH and CH), 13.17 (s, 1H, COOH). MS m/
z 587 (M þ 1).
CH2CH3), 0.84e1.02 (m, 2H, CH3CH2), 1.11 (d, 3H, J ¼ 6.3 Hz, CHCH3),
2.38e2.43 (m, 1H, CH3CH), 2.44 (s, 1H, CH3), 5.11 (d, 1H, J ¼ 7.8 Hz,
NCH), 6.86e8.02 (m, 11H, AreH and CH),13.20 (s, 1H, COOH). MS m/
z 508 (M þ 1).
5.7.8. (2S)-3-Methyl-2-((Z)-5-((3-methyl-5-(naphthalen-1-yloxy)-
1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-
3-yl)pentanoic acid (7h)
5.7.14. (2S)-3-Methyl-2-((Z)-5-((3-methyl-1-phenyl-5-(3-
(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)pentanoic acid (7n)
Yield 36%; m.p. 89e91 ꢀC. IR (KBr) cmꢁ1: 3424 (OH),1743 (C]O).
Yield 41%; m.p. 79e81 ꢀC. IR (KBr) cmꢁ1: 3425 (OH), 1754 (C]
1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.83 (t, 3H, J ¼ 7.9 Hz,
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.73 (t, 3H, J ¼ 7.8 Hz,
CH2CH3), 1.41 (d, 3H, J ¼ 6.4 Hz, CHCH3), 1.50e1.59 (m, 2H, CH3CH2),
2.42e2.48 (m, 1H, CH3CH), 2.49 (s, 1H, CH3), 4.72 (br.s, 1H, NCH),
6.06e8.51 (m, 13H, AreH and CH), 13.26 (s, 1H, COOH). MS m/z 558
(M þ 1).
CH2CH3), 0.89e1.07 (m, 2H, CH3CH2), 1.11 (d, 3H, J ¼ 6.2 Hz, CHCH3),
2.39e2.45 (m, 1H, CH3CH), 2.46 (s, 1H, CH3), 5.15 (d, 1H, J ¼ 8.3 Hz,
NCH), 7.19e7.60 (m, 10H, AreH and CH), 13.15 (s,1H, COOH). MS m/z
576 (M þ 1).
5.7.9. (2S)-2-((Z)-5-((5-(2,4-Dimethylphenoxy)-3-methyl-1-
phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)-3-methylpentanoic acid (7i)
5.7.15. (2S)-2-((Z)-5-((5-(4-Methoxyphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylpentanoic acid (7o)
Yield 44%; m.p. 188e190 ꢀC. IR (KBr) cmꢁ1: 3420 (OH), 1744
Yield 41%; m.p. 124e126 ꢀC. IR (KBr) cmꢁ1: 3414 (OH), 1747 (C]
(C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 0.74 (t, 3H,
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.75 (t, 3H, J ¼ 7.6 Hz,
J ¼ 7.9 Hz, CH2CH3), 1.11 (d, 3H, J ¼ 6.6 Hz, CHCH3), 1.15e1.26 (m,
2H, CH3CH2), 2.11 (s, 1H, ph-CH3), 2.36 (s, 1H, ph-CH3), 2.38e2.43
(m, 1H, CH3CH), 2.44 (s, 1H, CH3), 5.15 (d, 1H, J ¼ 8.1 Hz, NCH),
6.25e7.54 (m, 9H, AreH and CH), 13.17 (s, 1H, COOH). MS m/z 536
(M þ 1).
CH2CH3), 0.90e1.04 (m, 2H, CH3CH2), 1.11 (d, 3H, J ¼ 6.4 Hz, CHCH3),
2.37e2.43 (m, 1H, CH3CH), 2.44 (s, 1H, CH3), 3.73 (s, 1H, ph-CH3),
5.16 (d, 1H, J ¼ 7.4 Hz, NCH), 6.81e7.61 (m, 10H, AreH and CH), 13.16
(s, 1H, COOH). MS m/z 538 (M þ 1).
5.7.16. (2S)-2-((Z)-5-((5-(2-Methoxyphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
methylpentanoic acid (7p)
5.7.10. (2S)-3-Methyl-2-((Z)-5-((3-methyl-1-phenyl-5-(o-
tolyloxy)-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)pentanoic acid (7j)
Yield 38%; m.p. 109e111 ꢀC. IR (KBr) cmꢁ1: 3419 (OH), 1752 (C]
Yield 56%; m.p. 185e187 ꢀC. IR (KBr) cmꢁ1: 3409 (OH), 1738 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.84 (t, 3H, J ¼ 7.9 Hz,
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.75 (t, 3H, J ¼ 7.8 Hz,
CH2CH3), 0.94e1.08 (m, 2H, CH3CH2),1.23 (d, 3H, J ¼ 6.3 Hz, CHCH3),
2.33e2.40 (m, 1H, CH3CH), 2.41 (s, 1H, CH3), 3.73 (s, 1H, ph-CH3),
4.82 (br.s, 1H, NCH), 6.57e7.57 (m, 10H, AreH and CH), 12.92 (s, 1H,
COOH). MS m/z 538 (M þ 1).
CH2CH3), 1.11 (d, 3H, J ¼ 6.5 Hz, CHCH3), 1.21e1.29 (m, 2H, CH3CH2),
2.40 (s, 1H, ph-CH3), 2.41e2.44 (m, 1H, CH3CH), 2.45 (s, 1H, CH3),
5.14 (d,1H, J ¼ 8.9 Hz, NCH), 6.38e7.55 (m,10H, AreH and CH),13.15
(s, 1H, COOH). MS m/z 522 (M þ 1).
5.7.11. (2S)-3-Methyl-2-((Z)-5-((3-methyl-1-phenyl-5-(m-
tolyloxy)-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)pentanoic acid (7k)
5.8. General procedure for the preparation of compound 8
A mixture of appropriate 5-(substituted phenoxy)-3-methyl-1-
phenyl-1H-pyrazole-4-carbaldehyde (3 mmol), (S)-4-methyl-2-(4-
oxo-2-thioxothiazolidin-3-yl)pentanoic acid (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, 80:1) to get
a yellow solid.
Yield 53%; m.p. 184e186 ꢀC. IR (KBr) cmꢁ1: 3420 (OH), 1752
(C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.75 (t, 3H,
J ¼ 7.6 Hz, CH2CH3), 0.80e0.91 (m, 2H, CH3CH2), 1.12 (d, 3H,
6.3 Hz, CHCH3), 2.16 (s, 1H, ph-CH3), 2.40e2.44
J
¼
(m, 1H, CH3CH), 2.45 (s, 1H, CH3), 5.16 (br.s, 1H, NCH), 6.75e
7.61 (m, 10H, AreH and CH), 13.17 (s, 1H, COOH). MS m/z 522
(M þ 1).

384
M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
5.8.1. (S,Z)-2-(5-((5-(2,4-Dichlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-
methylpentanoic acid (8a)
3.02 (m, 2H, CHCH2), 5.49 (br.s, 1H, CH2CH), 6.67e7.73 (m, 9H,
AreH and CH), 10.75 (s, 1H, NH), 13.22 (s, 2H, COOH). MS m/z 579
(M þ 1).
Yield 35%; m.p. 120e122 ꢀC. IR (KBr) cmꢁ1: 3425 (OH), 1718 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.80 (d, 3H, J ¼ 6.51 Hz,
5.11. General procedure for the preparation of compound 11
CHCH3), 0.85 (d, 3H, J ¼ 6. 45 Hz, CHCH3), 1.15 (m, 1H, CHCH2-Ha),
1.37 (m, 1H, CHCH2-Hb), 2.14 (m, 1H, CH (CH3)2), 2.44 (s, 3H, CH3),
5.51 (br.s, 1H, NCH), 7.17e7.71 (m, 9H, AreH and CH), 13.27 (s, 1H,
COOH). MS m/z 576 (M þ 1).
A mixture of appropriate 5-(substituted phenoxy)-3-methyl-1-
phenyl-1H-pyrazole-4-carbaldehyde (3 mmol), (2S)-3-hydroxy-2-
(4-oxo-2-thioxothiazolidin-3-yl)butanoic acid (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/acetic acid,
100:1:0.5) to get a yellow solid.
5.8.2. (S,Z)-2-(5-((5-(4-Bromophenoxy)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-
methylpentanoic acid (8b)
Yield 42%; m.p. 98e100 ꢀC. IR (KBr) cmꢁ1: 3426 (OH), 1717 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.80 (d, 3H, J ¼ 6.51 Hz,
CHCH3), 0.86 (d, 3H, J ¼ 6. 36 Hz, CHCH3), 1.36 (m, 1H, CHCH2-Ha),
1.97 (m, 1H, CHCH2-Hb), 2.12 (m, 1H, CH (CH3)2), 2.44 (s, 3H, CH3),
5.51 (br.s, 1H, NCH), 7.29e7.59 (m, 10H, AreH and CH), 13.30 (s, 1H,
COOH). MS m/z 587 (M þ 1).
5.11.1. (2S)-2-((Z)-5-((5-(2,4-Dichlorophenoxy)-3-methyl-1-
phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)-3-hydroxybutanoic acid (11a)
Yield 23%; m.p. 107e109 ꢀC. IR (KBr) cmꢁ1: 3414, 2924 (OH),
1674 (C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 1.33 (d, 3H,
5.8.3. (S,Z)-2-(5-((5-(2,4-Dimethylphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-
methylpentanoic acid (8c)
J ¼ 6.0 Hz, CHCH3), 2.44 (s, 3H, CH3), 4.26e4.33 (m, 1H, CH3CH),
5.02 (br.s, 1H, NCH), 6.66e7.56 (m, 10H, AreH), 7.72 (s, 1H, OH),
13.14 (s, 1H, COOH). MS m/z 565 (M þ 1).
Yield 32%; m.p. 81e83 ꢀC. IR (KBr) cmꢁ1: 3429 (OH),1713 (C]O).
1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.82 (d, 3H, J ¼ 6.36 Hz,
5.11.2. (2S)-2-((Z)-5-((5-(2,4-Dimethylphenoxy)-3-methyl-1-
phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)-3-hydroxybutanoic acid (11b)
CHCH3), 0.87 (d, 3H, J ¼ 6.54 Hz, CHCH3), 1.24 (m, 1H, CHCH2-Ha),
1.37 (m, 1H, CHCH2-Hb), 1.96 (m, 1H, CH (CH3)2), 2.11 (s, 3H, CH3),
2.37 (s, 3H, ph-CH3), 2.45 (s, 3H, ph-CH3), 5.51 (br.s, 1H, NCH), 6.23e
7.66 (m, 9H, AreH and CH),13.35 (s,1H, COOH). MS m/z 536 (M þ 1).
Yield 19%; m.p.110e112 ꢀC. IR (KBr) cmꢁ1: 3412, 2921 (OH),1676
(C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
CHCH3), 2.11 (s, 3H, ph-CH3), 2.38 (s, 3H, ph-CH3), 2.44 (s, 3H, CH3),
4.32e4.41 (m, 1H, CH3CH), 5.05 (br.s, 1H, NCH), 7.01 (s, 1H, OH),
6.76e7.60 (m, 9H, AreH), 13.13 (s, 1H, COOH). 13C NMR (DMSO-d6,
d
1.34 (d, 3H, J ¼ 6.1 Hz,
5.9. General procedure for the preparation of compound 9
A mixture of 5-(2,4-dimethylphenoxy)-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde (3 mmol), (S)-2-(4-oxo-2-thioxothiazolidin-
3-yl)pentanedioic acid (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/acetic acid, 50:1:0.5) to get a yellow
solid.
300 MHz, ppm): d 186.17, 152.27, 150.36, 150.18, 145.32, 138.31,
136.57, 132.68, 132.33, 129.23, 128.99, 128.82, 127.97, 127.68, 125.95,
125.03, 122.42,112.17, 105.15, 43.39, 21.72,19.96, 15.83, 13.09. MS m/
z 524 (M þ 1).
5.12. General procedure for the preparation of compound 12
A mixture of appropriate 5-(substituted phenoxy)-3-methyl-1-
phenyl-1H-pyrazole-4-carbaldehyde (3 mmol), (S)-3-hydroxy-2-
(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid (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/acetic acid,
100:1:0.5) to get a yellow solid.
5.9.1. (S,Z)-2-(5-((5-(2,4-Dimethylphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)
pentanedioic acid (9a)
Yield 20%; m.p. 144e146 ꢀC. IR (KBr) cmꢁ1: 3406 (OH), 1713 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.11 (s, 3H, ph-CH3), 2.17e
3.23 (m, 2H, CHCH2), 2.36 (s, 3H, ph-CH3), 2.44 (s, 3H, CH3), 3.36e
3.43 (m, 2H, COCH2), 5.28 (br.s, 1H, NCH), 6.21e7.55 (m, 9H, AreH
and CH), 13.18 (s, 2H, COOH). MS m/z 552 (M þ 1).
5.12.1. (S,Z)-2-(5-((5-(2,4-Dichlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
hydroxypropanoic acid (12a)
5.10. The procedure for the preparation of compound 10
Yield 21%; m.p. 191e193 ꢀC. IR (KBr) cmꢁ1: 3448, 2904 (OH),
A mixture of 5-(2,4-dichlorophenoxy)-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde (3 mmol), (S)-2-(4-oxo-2-thioxothiazolidin-
3-yl)succinic acid (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 (dichloro-
methane/methanol/acetic acid, 50:1:0.5) to get a yellow solid.
1703 (C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.45 (s, 3H,
CH3), 4.01e4.11 (m, 2H, CHCH2), 5.46 (s, 1H, CH2CH), 5.76 (s, 1H,
CH2OH), 6.67e7.72 (m, 9H, AreH), 13.24 (s, 1H, COOH). MS m/z 551
(M þ 1).
5.12.2. (S,Z)-2-(5-((5-(2,4-Dimethylphenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
hydroxypropanoic acid (12b)
5.10.1. (S,Z)-2-(5-((5-(2,4-Dichlorophenoxy)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)
succinic acid (10a)
Yield 19%; m.p. 199e201 ꢀC. IR (KBr) cmꢁ1: 3453, 2901 (OH),
1709 (C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 1.99 (s, 3H, ph-
CH3), 2.38 (s, 3H, ph-CH3), 2.45 (s, 3H, CH3), 4.01e4.10 (m, 2H,
CHCH2), 5.50 (s, 1H, CH2CH), 5.76 (s, 1H, CH2OH), 6.21e7.56 (m, 9H,
AreH), 13.21 (s, 1H, COOH). 13C NMR (DMSO-d6, 300 MHz, ppm):
Yield 23%; m.p. 131e133 ꢀC. IR (KBr) cmꢁ1: 3406 (OH), 1713 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.45 (s, 3H, CH3), 2.94e

M.-X. Song et al. / European Journal of Medicinal Chemistry 60 (2013) 376e385
385
d
186.17, 152.27, 150.36, 150.18, 145.32, 138.31, 136.57, 132.68,
prepared in DMSO, the final concentration of which did not exceed
132.33, 129.23, 128.99, 128.82, 127.97, 127.68, 125.95, 125.03,
122.42, 112.17, 105.15, 43.39, 21.72, 19.96, 15.83, 13.09. MS m/z 510
(M þ 1).
0.05%. A two-fold serial dilution technique [13] was used to obtain
final concentrations of 64e0.5 mg/mL. The MIC was defined as the
concentration of a test compound that completely inhibited
bacteria growth during 24 h incubation at 37 ꢀC. Bacteria growth
was determined by measuring the absorption at 650 nm using
a microtiter enzyme-linked immunosorbent assay (ELISA) reader.
All experiments were carried out three times.
5.13. General procedure for the preparation of compound 13
A mixture of compound 3 (3 mmol), appropriate rhodanines
(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 get a yellow solid.
5.15. Evaluation of cytotoxicity in vitro
Human cervical (HeLa) cell monolayers were used as an in vitro
model of cervicovaginal epithelium for testing the cytotoxicity of
the new compounds. HeLa cells were grown in Dulbecco modified
Eagle medium supplemented with fetal bovine serum (10%), and
antibiotics (penicillinestreptomycin mixture [100 U/mL]). Cells at
80e90% confluence were split by trypsin (0.25% in PBS; pH 7.4), and
the medium was changed at 24 h intervals. The cells were cultured
at 37 ꢀC in a 5% CO2 incubator. The cells were grown to 3 passages
and approximately 1 ꢂ104 cells were seeded into each well of a 96-
well plate and allowed to incubate overnight to allow cells to attach
to the substrate. After 24 h, the medium was replaced with DMEM
supplemented with 10% FBS containing various concentrations of
5.13.1. (Z)-2-(5-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (13a)
Yield 76%; m.p. 146e148 ꢀC. IR (KBr) cmꢁ1: 3427 (OH), 1709 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d 2.46 (s, 3H, CH3), 4.54 (s,
2H, NCH2), 7.45e7.73 (m, 6H, AreH and CH), 13.40 (s, 1H, COOH).
MS m/z 395 (M þ 1).
5.13.2. (S,Z)-2-(5-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-methylbutanoic acid
(13b)
test compounds and incubated for 48 h. Then 10 ml of MTT solution
(5 mg/mL in PBS) was added to each well. After incubation for 4 h,
the medium was removed and the resulting formazan crystals were
Yield 43%; m.p. 112e114 ꢀC. IR (KBr) cmꢁ1: 3421 (OH), 1704 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.78 (d, 3H, J ¼ 6.72 Hz,
CHCH3), 0.93 (m, 1H, CH3CH), 1.26 (d, 3H, J ¼ 6.36 Hz, CHCH3), 2.44
(s, 3H, CH3), 5.13 (br.s,1H, CH2CH), 7.44e8.02 (m, 6H, AreH and CH),
13.71 (s, 1H, COOH). MS m/z 436 (M þ 1).
dissolved with 100
ml DMSO. After shaking 10 min, the optical
density was measured at 570 nm using a microtiter ELISA reader.
The assay was conducted four times. The IC50 values were defined
as the concentrations inhibiting 50% of cell growth.
5.13.3. (2S)-2-((Z)-5-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-
yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-3-methylpentanoic
acid (13c)
Acknowledgment
Yield 50%; m.p. 106e108 ꢀC. IR (KBr) cmꢁ1: 3418 (OH), 1702 (C]
This work was supported by the National Science Foundation of
China (grant number 20962021 and 81260468).
O). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
0.85 (t, 3H, J ¼ 7.23 Hz,
CHCH3), 0.99 (m, 2H, CH3CH2), 1.20 (d, 3H, J ¼ 6.48 Hz, CHCH3), 2.45
(s, 3H, CH3), 5.27 (br.s,1H, CH2CH), 7.56e7.99 (m, 6H, AreH and CH),
13.30 (s, 1H, COOH). MS m/z 450 (M þ 1).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.007.
5.13.4. (S,Z)-2-(5-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic
acid (13d)
References
Yield 38%; m.p. 109e111 ꢀC. IR (KBr) cmꢁ1: 3423 (OH), 1715 (C]
O). 1H NMR (DMSO-d6, 300 MHz, ppm): 0.92 (t, 3H, J ¼ 6.57 Hz,
CHCH3), 0.97 (t, 3H, J ¼ 6.48 Hz, CHCH3), 5.63 (br.s, 1H, CH2CH),
7.56e7.67 (m, 6H, AreH and CH), 13.44 (s, 1H, COOH). MS m/z 436
(M þ 1).
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