Synthesis and biological activities of some new thiazolidine derivatives containing pyrazole ring system

Article abstract of DOI:10.1002/jhet.834

As a part of systematic investigation of synthesis and biologically active compounds of thiazolidine (TZD) derivatives containing pyrazole ring system, several new pyrazole-TZD derivatives 8a-d and 9a-d have been synthesized. Compounds 8a-d were prepared from N-substituted TZDs 6a-d and 1H-pyrazole-4-carboxaldehyde 7 by Knoevenagel-type reaction. Treatment of 8a-d with sodium hydride at room temperature caused dimerization reaction to afford the corresponding spirocompounds 9a-d. All the synthesized compounds were characterized by spectroscopic analysis. In vitro, the synthesized compounds 8a-d and 9a-d were tested for their growth inhibitory activity in A549 lung cancer, B16F10 murine melanoma, and HeLa human uterine carcinoma cells and for their differentiation of 3T3-L1 preadipocytes to adipocytes. The results showed that compound 8c possessed growth inhibitory effect of B16F10 cells (IC ₅₀ = 27 μM) and compounds 9c,d had induction effect on the differentiation of 3T3-L1 preadipocytes. Copyright

Full text of DOI:10.1002/jhet.834

March 2012
Synthesis and Biological Activities of Some New Thiazolidine
Derivatives Containing Pyrazole Ring System
303
Sho Nishida, Hiroshi Maruoka,* Yuki Yoshimura, Takao Goto, Ryoko Tomita,
Eiichi Masumoto, Fumi Okabe, Kenji Yamagata, and Toshihiro Fujioka
Laboratory of Instrumental Analysis, Faculty of Pharmaceutical Sciences, Fukuoka University,
Jonan-ku, Fukuoka 814-0180, Japan
*E-mail: maruoka@fukuoka-u.ac.jp
Received October 7, 2010
DOI 10.1002/jhet.834
Published online 21 November 2011 in Wiley Online Library (wileyonlinelibrary.com).
As a part of systematic investigation of synthesis and biologically active compounds of thiazolidine (TZD)
derivatives containing pyrazole ring system, several new pyrazole–TZD derivatives 8aꢀd and 9aꢀd have
been synthesized. Compounds 8aꢀd were prepared from N-substituted TZDs 6aꢀd and 1H-pyrazole-4-
carboxaldehyde 7 by Knoevenagel-type reaction. Treatment of 8aꢀd with sodium hydride at room tempera-
ture caused dimerization reaction to afford the corresponding spirocompounds 9aꢀd. All the synthesized
compounds were characterized by spectroscopic analysis. In vitro, the synthesized compounds 8aꢀd and
9aꢀd were tested for their growth inhibitory activity in A549 lung cancer, B16F10 murine melanoma, and
HeLa human uterine carcinoma cells and for their differentiation of 3T3-L1 preadipocytes to adipocytes. The
results showed that compound 8c possessed growth inhibitory effect of B16F10 cells (IC50 ¼ 27 lM) and
compounds 9c,d had induction effect on the differentiation of 3T3-L1 preadipocytes.
J. Heterocyclic Chem., 49, 303 (2012).
INTRODUCTION
On the other hand, compounds containing the pyraz-
ole ring system are known to possess pharmacological
activities such as analgesic, antidepressant, antibacterial,
plant growth regulatory, anti-inflammatory, and
antihyperglycemic activities [14–19]. Therefore, the de-
velopment of improved methods for the synthesis of
substituted pyrazole derivatives has acquired relevance
to current research [20–23].
The thiazolidine (TZD) and pyrazole derivatives are
the most extensively investigated classes of compounds.
TZDs classes of insulin sensitizers, synthesized in early
1980s [1], were later found to mediate hypoglycemic
effect through peroxisome proliferator-activated receptor
c (PPARc) [2]. Currently, rosiglitazone and pioglitazone
are clinically available TZDs for the treatment of type 2
diabetes. Furthermore, TZD derivatives have attracted
very significant biochemical interest, owing to the pres-
ence of the TZD moiety in the structures of several
naturally occurring molecules with important pharmaco-
logical properties such as antidiabetic, antibiotic, and
antifungal activities [3–7]. In this context, the synthesis
of TZD derivatives continues to attract attention and
provides an interesting challenge [8–13].
Based on these properties, it can be reasonably sup-
posed that the development of synthetic strategies for
new TZD derivatives containing the pyrazole ring sys-
tem might provide additional lead molecules for drug
discovery. The molecular hybridization assumption
encouraged us to design a specific programme aimed at
synthesizing several new derivatives of these ring sys-
tem. To the best of our knowledge, there are relatively
C
V
2011 HeteroCorporation

304
S. Nishida, H. Maruoka, Y. Yoshimura, T. Goto, R. Tomita, E. Masumoto,
F. Okabe, K. Yamagata, and T. Fujioka
Vol 49
Table 1
Synthesis of compounds 6a–d and 8a–d according to Scheme 1.
Entry
Product
Yield (%)
1
2
3
4
5
6
7
8
6a [3,5,37]
6b [34,36]
6c
6d [5]
8a
90
96
99
78
66
65
64
42
8b
8c
8d
Figure 1. Structures of reported TZD derivatives containing pyrazole
ring system.
of 6a–d, 7, and sodium acetate in the absence of solvent
was stirred at 140ꢁC for 30 min, the expected pyrazole–
TZD derivatives 8a–d were obtained in moderate yields
(Scheme 1 and entries 5–8 in Table 1). The infrared
(IR) spectra of 8a–d display bands in the range of
few methods on the literature describing the preparation
of pyrazole–TZD derivatives 1 [24], 2 [25], and 3 [9]
(Fig. 1), even though both TZDs [1,2,26–28] and pyra-
zoles [29–33] have antitumor and antidiabetical activ-
ities. For these reasons, we have been interested in the
preparation of various types of pyrazole–TZD deriva-
tives to evaluate their biological activities and now
report the results of our investigation, the growth inhibi-
tory and differentiation-inducing activities of some new
pyrazole–TZD derivatives in tumor and preadipose cells.
1
1651–1753 cmꢀ1 due to four carbonyl groups. The H
NMR spectra of 8a–d in deuteriochloroform exhibit a
two-proton singlet near d 4.4 attributable to the methyl-
ene protons and a one-proton singlet near d 7.6 due to
the olefin proton. The 13C NMR spectra of 8a–d show a
signal near d 42 due to the methylene carbon, a signal
near d 124 due to the olefin carbon, and four signals in
the range of d 162–170 due to the carbonyl carbons.
Interestingly, the NMR spectra indicated that 8a–d
existed as a geometrical single isomer of Z configura-
tion. It seems that the exocyclic double bond is
exclusively in the Z configuration because of the high
degree of thermodynamic stability of this isomer
[28,40]. Elemental analyses and spectral data of 8a–d
are consistent with the proposed structures (see Experi-
mental section).
RESULTS AND DISCUSSION
Initially, the synthesis of N-substituted TZDs 6a–d
has been accomplished as outlined in Scheme 1. In fact,
2,4-thiazolidinedione (4) reacted smoothly with a-bromo
esters 5a–d in the presence of sodium hydride in reflux-
ing tetrahydrofuran to give the corresponding 6a–d
[3,5,34–37] in good yields. The results are summarized
in Table 1 (entries 1–4). Elemental analyses and spectral
data of 6a–d are consistent with the assigned structures
(see Experimental section).
Based on these results, we have tried to directly con-
struct pyrazole–TZD derivatives 8a–d from 4, a-bromo
esters 5a–d and 1H-pyrazole-4-carboxaldehyde 7 in a
one-pot process. The results are summarized in Table 2.
Thus, after a mixture of 4 and 1.0 equivalent of 5a–d in
the presence of 1.0 equivalent of sodium hydride in tet-
rahydrofuran was refluxed for 3 h, the reaction mixture
was treated with 1.0 equivalent of 1H-pyrazole-4-
carboxaldehyde 7 in the presence of 1.0 equivalent of
In the next step, a Knoevenagel-type condensation
[3,5,7,38,39] of N-substituted TZDs 6a–d and 1H-pyraz-
ole-4-carboxaldehyde 7 was examined. When a mixture
Scheme 1
Table 2
One-pot synthesis of pyrazole–thiazolindine derivatives 8a–d.
1Þ NaH; 5aꢀd: THF
4ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ! 8a ꢀ d
2Þ 7; AcONa
Entry
Substrate
aꢀBromo Esters
Product
Yield (%)
1
2
3
4
4
4
4
4
5a
5b
5c
5d
8a
8b
8c
8d
54
52
60
41
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DOI 10.1002/jhet

March 2012
Synthesis and Biological Activities of Some New Thiazolidine
Derivatives Containing Pyrazole Ring System
305
Scheme 2
The formation of the spirocompounds 9a–d could be
explained by possible mechanism presented in Scheme 2.
An isomerization of the keto form 8a–d to the enol
form A would easily occur in the presence of sodium
hydride. Thus, the reaction of 8a–d with sodium hydride
probably causes the conjugate addition of A to 8a–d to
give the intermediate Michael adducts B, which could
then undergo intramolecular conjugate addition to afford
the corresponding spirocompounds 9a–d. In this reac-
tion, the intermediate Michael adducts B were not be
detected. It makes us believe that the intermolecular/
intramolecular Michael addition reaction, namely dime-
rization reaction of 8a–d can readily be promoted by
using a sodium hydride/N,N-dimethylformamide system.
To elucidate in vitro biological activity of 8a–d and
9a–d, their antitumor activity was measured in A549
lung cancer, B16F10 murine melanoma, and HeLa
human uterine carcinoma cells using MTT assay
[41,42]. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) cell proliferation assay
has been widely accepted as a reliable way to measure
the cell proliferation rate. Although the data obtained by
MTT assay showed that almost the synthesized com-
pounds had no inhibitory effects on the growth of tumor
cells in dosage-dependent manners, compound 8c was
the most potent compound in this series, having a
growth inhibitory property (IC50) value of 27 lM in
suppressing B16F10 cell growth.
sodium acetate at 140ꢁC for 30 min without solvent, the
desired pyrazole–TZD derivatives 8a–d were obtained
in moderate yields (entries 1–4).
Furthermore, the differentiation-inducing effect of
compounds 8a–d and 9a–d on preadipose cells was also
investigated using Oil Red O-staining to assess triglycer-
ide accumulation in 3T3-L1 cells [43], which were cul-
tured with compounds 8a–d and 9a–d. The results are
shown in Figure 2. Pioglitazone, as a full PPARc ago-
nist, was used as a positive control and the absorbance
value in its group was significantly larger than that in
the induced medium. It is worth noting that compounds
9c,d were shown to possess high differentiation-inducing
activity in this pyrazole-containing TZD series.
Interestingly, we found the reaction condition under
which the spirocompounds 9a–d could be isolated in the
presence of sodium hydride. Subsequently, treatment of
pyrazole–TZD derivatives 8a–d with sodium hydride in
N,N-dimethylformamide at room temperature caused
dimerization reaction to give the corresponding spiro-
compounds 9a–d in moderate yields (Scheme 2 and
Table 3). The IR spectra of 9a–d display bands in the
range of 1649ꢀ1755 cmꢀ1 due to eight carbonyl groups.
The 1H NMR spectra of 9a–d in deuteriochloroform
exhibit a three-proton singlet near d 2.2 attributable to
the methyl protons and two three-proton singlets near d
3.1 due to two N-methyl groups. The 13C NMR spectra
of 9a–d show a signal near d 12 due to the methyl car-
bon, two signals near d 35 due to two N-methyl carbons,
three signals in the range of d 39–50 due to the methine
carbon, a signal near d 70 due to the spirocarbon, and
eight signals in the range of d 163–175 due to the car-
bonyl carbon. By comparison of the NMR, mass spectra,
and elemental analyses of 9a–d, it seems that the struc-
tural assignments given to these compounds are correct
(see Experimental section). Although there are several
possible stereoisomers 9a–d, the configuration of isolated
compounds was not confirmed by NMR spectroscopy.
In conclusion, we have described a facile approach to
prepare pyrazole–TZD derivatives 8a–d by a Knoevena-
gel-type condensation of N-substituted TZDs 6a–d and
1H-pyrazole-4-carboxaldehyde 7. Furthermore, we have
developed a novel method for the construction of spiro-
compounds 9a–d, proceeding by a dimerization reaction
Table 3
Reaction of compounds 8a–d with sodium hydride.
Entry
Substrate
Product
Yield (%)
1
2
3
4
8a
8b
8c
8d
9a
9b
9c
9d
50
28
41
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S. Nishida, H. Maruoka, Y. Yoshimura, T. Goto, R. Tomita, E. Masumoto,
F. Okabe, K. Yamagata, and T. Fujioka
Vol 49
ceased. To the obtained solution was added methyl bro-
moacetate (5a) (2.29 g, 15 mmol), ethyl bromoacetate
(5b) (2.51 g, 15 mmol), isopropyl bromoacetate (5c)
(2.72 g, 15 mmol), or tert-butyl bromoacetate (5d) (2.93
g, 15 mmol) with stirring and ice cooling, and then the
mixture was refluxed for 3 h. After removal of the sol-
vent in vacuo, cold water was added to the residue. The
resulting mixture was extracted with ethyl acetate (60
mL). The extract was dried over anhydrous sodium sul-
fate and concentrated in vacuo. The residue was purified
by column chromatography on silica gel with chloro-
form as the eluent to give 6a [3,5,35] (1.71 g, 90%), 6b
[32,34] (1.95 g, 96%), 6c (2.14 g, 99%), and 6d [5]
(1.81 g, 78%).
Figure 2. Induction effect of compounds 8a–d and 9a–d on differen-
tiation of 3T3-L1 to adipocytes. The data were presented as mean 6
SE (n ¼ 8). **, p < 0.01, when compared with induced medium; N,
noninduced medium; I, induced medium; P, pioglitazone.
Isopropyl (2,4-dioxo-3-thiazolidine)acetate (6c). This
compound was obtained as pale yellow oil; IR (neat): m
1742, 1690 cmꢀ1 (C¼¼O); 1H NMR (deuteriochloro-
form): d 1.26 (d, J ¼ 6.1 Hz, 6H, CO2CHMe2), 4.03 (s,
2H, TZD 5-H), 4.30 (s, 2H, a-CH2), 5.06 ppm (sep, J ¼
6.1 Hz, 1H, CO2CHMe2); 13C NMR (deuteriochloro-
form): d 21.7 (CO2CHMe2), 33.8 (TZD C-5), 42.4 (a-
CH2), 70.1 (CO2CHMe2), 165.6 (CO2CHMe2), 170.1
(TZD C-4), 171.0 ppm (TZD C-2); ms: m/z 218
[MþH]þ. Anal. Calcd. for C8H11NO4S: C, 44.23; H,
5.10; N, 6.45. Found: C, 44.16; H, 5.12; N, 6.19.
General procedure for the preparation of pyrazole–
TZDs 8a–d from 6a–d and 7. A mixture of 6a–d (10
mmol), 7 (2.16 g, 10 mmol), and sodium acetate (0.82
g, 10 mmol) was stirred at 140ꢁC for 30 min. The
reaction mixture was purified by column chromatography
on silica gel with chloroform as the eluent to afford
8a–d.
when 8a–d were treated with sodium hydride. Interest-
ingly, we found that compound 8c could suppress
B16F10 cancer cell growth. 8c was the most effective
molecule in inhibiting B16F10 cell growth and might
perform its action through including apoptosis. In addi-
tion, compounds 9c,d showed high differentiation-induc-
ing activity in 3T3-L1 preadipocytes in vitro, which was
similar in effect to that of pioglitazone as a PPARc ago-
nist. Our results suggest that these new pyrazole–TZD
derivatives might play a role in vivo as anticancer or
antidiabetic agents. Functionalized pyrazole–TZD deriv-
atives are important synthons in organic synthesis and
for the preparation of biologically active compounds
with interest in medicinal chemistry. Further studies on
the synthesis of new substituted pyrazole–TZD deriva-
tives are under way.
Methyl (Z)-{5-[(1,2-dihydro-1,5-dimethyl-3-oxo-2-
phenyl-3H-pyrazol-4-yl)methylene]-2,4-dioxo-3-thia-
zolidine}acetate (8a). This compound was obtained as
pale yellow needles, mp 201–203ꢁC (chloroform–petro-
leum ether); IR (potassium bromide): m 1742, 1716, 1670
EXPERIMENTAL
1
All melting points are uncorrected. The IR spectra
were recorded on a JASCO Fourier transform infrared
cmꢀ1 (C¼¼O); H NMR (deuteriochloroform): d 2.40 (s,
3H, pyrazole 5-Me), 3.30 (s, 3H, pyrazole 1-Me), 3.75 (s,
3H, CO2Me), 4.43 (s, 2H, NCH2), 7.31–7.33 (m, 2H, Ph-
H), 7.38–7.41 (m, 1H, Ph-H), 7.48–7.51 (m, 2H, Ph-H),
7.59 ppm (s, 1H, C¼¼CHA); 13C NMR (deuteriochloro-
form): d 10.9 (pyrazole 5-Me), 34.7 (pyrazole 1-Me),
41.7 (NCH2), 52.6 (CO2Me), 102.7 (pyrazole C-4), 117.0
(TZD C-5), 124.4 (C¼¼CHA), 126.1, 128.5, 129.6, 133.5
(Ph-C), 153.4 (pyrazole C-5), 162.3 (pyrazole C-3),
166.8, 167.1 (2 ꢂ C¼¼O), 169.8 ppm (CO2Me); ms: m/z
388 [MþH]þ. Anal. Calcd. for C18H17N3O5S: C, 55.80;
H, 4.42; N, 10.85. Found: C, 55.52; H, 4.49; N, 10.75.
1
spectroscopy (FT/IR)-4100 spectrometer. The H NMR
and 13C NMR spectra were recorded on a JEOL JNM-
A500 spectrometer at 500 and 125 MHz, respectively.
1
The H and 13C chemical shifts (d) are reported in parts
per million (ppm) relative to tetramethylsilane as inter-
nal standard. The positive fast atom bombardment
(FAB) mass spectra were obtained on a JEOL JMS-
700T spectrometer. The elemental analyses were per-
formed on a YANACO MT-6 CHN analyzer.
General procedure for the preparation of N-
substituted TZDs 6a–d from 4 and a-bromo esters
5a–d. To an ice-cooled and stirred solution of 4 (1.17
g, 10 mmol) in tetrahydrofuran (30 mL) was added 60%
sodium hydride (0.44 g, 11 mmol). The stirring was
continued at room temperature until evolution of gas
Ethyl
(Z)-{5-[(1,2-dihydro-1,5-dimethyl-3-oxo-2-
phenyl-3H-pyrazol-4-yl)methylene]-2,4-dioxo-3-thia-
zolidine}acetate (8b). This compound was obtained as
pale yellow needles, mp 165–167ꢁC (acetone–petroleum
ether); IR (potassium bromide): m 1747, 1727, 1672,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

March 2012
Synthesis and Biological Activities of Some New Thiazolidine
Derivatives Containing Pyrazole Ring System
307
1655 cmꢀ1 (C¼¼O); 1H NMR (deuteriochloroform): d
1.27 (t, J ¼ 7.0 Hz, 3H, CO2CH2Me), 2.40 (s, 3H, pyr-
azole 5-Me), 3.30 (s, 3H, pyrazole 1-Me), 4.21 (q, J ¼
7.0 Hz, 2H, CO2CH2Me), 4.41 (s, 2H, NCH2), 7.31–
7.33 (m, 2H, Ph-H), 7.38–7.41 (m, 1H, Ph-H), 7.48–
7.51 (m, 2H, Ph-H), 7.60 ppm (s, 1H, C¼¼CHA); 13C
NMR (deuteriochloroform): d 11.0 (pyrazole 5-Me),
14.1 (CO2CH2Me), 34.7 (pyrazole 1-Me), 41.9 (NCH2),
61.8 (CO2CH2Me), 102.8 (pyrazole C-4), 117.2 (TZD
C-5), 124.2 (C¼¼CHA), 126.1, 128.5, 129.6, 133.5 (Ph-
C), 153.5 (pyrazole C-5), 162.4 (pyrazole C-3), 166.6
(CO2CH2Me), 166.9, 169.8 ppm (2 ꢂ C¼¼O); ms: m/z
402 [MþH]þ. Anal. Calcd. for C19H19N3O5S: C, 56.85;
H, 4.77; N, 10.47. Found: C, 56.87; H, 4.79; N, 10.47.
Isopropyl (Z)-{5-[(1,2-dihydro-1,5-dimethyl-3-oxo-
2-phenyl-3H-pyrazol-4-yl)methylene]-2,4-dioxo-3-thia-
zolidine}acetate (8c). This compound was obtained as
pale yellow needles, mp 202–204ꢁC (chloroform–petro-
leum ether); IR (potassium bromide): m 1753, 1719, 1671,
1651 cmꢀ1 (C¼¼O); 1H NMR (deuteriochloroform): d
1.25 (d, J ¼ 6.4 Hz, 6H, CO2CHMe2), 2.39 (s, 3H, pyraz-
ole 5-Me), 3.30 (s, 3H, pyrazole 1-Me), 4.38 (s, 2H,
NCH2), 5.06 (sep, J ¼ 6.4 Hz, 1H, CO2CHMe2), 7.30–
7.32 (m, 2H, Ph-H), 7.37–7.41 (m, 1H, Ph-H), 7.47–7.51
(m, 2H, Ph-H), 7.59 ppm (s, 1H, C¼¼CHA); 13C NMR
(deuteriochloroform): d 10.9 (pyrazole 5-Me), 21.7
(CO2CHMe2), 34.7 (pyrazole 1-Me), 42.1 (NCH2), 69.7
(CO2CHMe2), 102.7 (pyrazole C-4), 117.1 (TZD C-5),
124.2 (C¼¼CHA), 126.1, 128.5, 129.6, 133.5 (Ph-C),
153.5 (pyrazole C-5), 162.3 (pyrazole C-3), 166.1
(CO2CHMe2), 166.9, 169.8 ppm (2 ꢂ C¼¼O); ms: m/z 416
[MþH]þ. Anal. Calcd. for C20H21N3O5S: C, 57.82; H,
5.09; N, 10.11. Found: C, 57.65; H, 5.15; N, 10.06.
drofuran (30 mL) was added 60% sodium hydride (0.40
g, 10 mmol). The stirring was continued at room tem-
perature until evolution of gas ceased. To the obtained
solution was added methyl bromoacetate (5a) (1.53 g,
10 mmol), ethyl bromoacetate (5b) (1.67 g, 10 mmol),
isopropyl bromoacetate (5c) (1.81 g, 10 mmol), or tert-
butyl bromoacetate (5d) (1.95 g, 10 mmol) with stirring
and ice cooling, and then the mixture was refluxed for 3
h. After removal of the deposited crystals by filtration,
the filtrate was concentrated in vacuo. To the obtained
residue was added 7 (2.16 g, 10 mmol) and sodium ace-
tate (0.82 g, 10 mmol), and then the mixture was stirred
at 140ꢁC for 30 min. The resulting mixture was purified
by column chromatography on silica gel with chloro-
form as the eluent to yield 8a (2.09 g, 54%), 8b (2.08 g,
52%), 8c (2.50 g, 60%), and 8d (1.76 g, 41%), respec-
tively. The melting points and IR spectra of 8a–d coin-
cided with those of authentic samples prepared from
6a–d and 7.
General procedure for the preparation of spiro-
compounds 9a–d from 8a–d and sodium hydride. To
an ice-cooled and stirred solution of 8a–d (1 mmol) in
N,N-dimethylformamide (10 mL) was added 60%
sodium hydride (0.08 g, 2 mmol). After the mixture was
stirred at room temperature overnight, a 5% hydrochlo-
ric acid solution (20 mL) was added to the reaction mix-
ture with stirring and ice cooling. The resulting mixture
was extracted with chloroform (60 mL). The extract was
dried over anhydrous sodium sulfate and concentrated in
vacuo. The residue was purified by column chromatography
on silica gel with chloroform–acetone (4:1) as the eluent
to give 9a–d.
10,40,60,70-Tetrahydro-60-(1,2-dihydro-1,5-dimethyl-
3-oxo-2-phenyl-3H-pyrazol-4-yl)-10-methyl-40-[(2,4-
dioxo-3-thiazolidine)acetic acid methyl ester-5-
yl]spiro[thiazolidine-5,50-[5H]indazol]-2,30,4(20H)-trione-
3-acetic acid methyl ester (9a).. This compound was
obtained as colorless needles, mp 175–177ꢁC dec. (ace-
tone–petroleum ether); IR (potassium bromide): m 1755,
tert-Butyl (Z)-{5-[(1,2-dihydro-1,5-dimethyl-3-oxo-
2-phenyl-3H-pyrazol-4-yl)methylene]-2,4-dioxo-3-thia-
zolidine}acetate (8d). This compound was obtained as
pale yellow needles, mp 191–193ꢁC dec. (chloroform–
petroleum ether); IR (potassium bromide): m 1739, 1719,
1671 cmꢀ1 (C¼¼O); 1H NMR (deuteriochloroform): d
1.46 (s, 9H, CO2CMe3), 2.40 (s, 3H, pyrazole 5-Me),
3.30 (s, 3H, pyrazole 1-Me), 4.32 (s, 2H, NCH2), 7.31–
7.33 (m, 2H, Ph-H), 7.37–7.41 (m, 1H, Ph-H), 7.48–
7.51 (m, 2H, Ph-H), 7.59 ppm (s, 1H, C¼¼CHA); 13C
NMR (deuteriochloroform): d 11.0 (pyrazole 5-Me),
28.0 (CO2CMe3), 34.7 (pyrazole 1-Me), 42.6 (NCH2),
82.7 (CO2CMe3), 102.9 (pyrazole C-4), 117.4 (TZD C-
5), 124.0 (C¼¼CHA), 126.0, 128.4, 130.0, 133.6 (Ph-C),
153.5 (pyrazole C-5), 162.4 (pyrazole C-3), 165.6, 167.0
(2 ꢂ C¼¼O), 170.0 ppm (CO2CMe2); ms: m/z 430
[MþH]þ. Anal. Calcd. for C21H23N3O5S: C, 58.73; H,
5.40; N, 9.78. Found: C, 58.75; H, 5.42; N, 9.62.
1
1733, 1702, 1682, 1649 cmꢀ1 (C¼¼O); H NMR (deuter-
iochloroform): d 2.18 (s, 3H, pyrazole 5-Me), 2.59–2.63
(m, 1H, 70-H), 3.01, 3.15 (s, 6H, 2 ꢂ pyrazole 1-Me),
3.72, 3.73 (s, 6H, 2 ꢂ CO2Me), 3.82 (dd, J ¼ 5.2, 12.9
Hz, 1H, 60-H), 4.30–4.32 (m, 2H, 40- and 70-H), 4.34,
4.42 (s, 4H, 2 ꢂ NCH2), 4.84 (s, 1H, TZD 5-H), 7.24–
7.35 (m, 6H, Ph-H), 7.39–7.46 ppm (m, 4H, Ph-H); 13C
NMR (deuteriochloroform): d 11.6 (pyrazole 5-Me),
23.3 (C-70), 35.3, 35.5 (2 ꢂ pyrazole 1-Me), 39.7 (C-60),
42.0, 42.3 (2 ꢂ NCH2), 44.0 (TZD C-5), 49.6 (C-40),
52.4, 52.7 (2 ꢂ CO2Me), 69.8 (C-50), 101.0 (pyrazole
C-4), 102.5 (C-30a), 124.5, 125.0, 127.0, 127.2, 129.17,
129.2, 134.4, 134.6, (Ph-C), 154.5 (pyrazole C-5), 154.8
(C-70a), 163.4 (C-30), 164.8 (pyrazole C-3), 166.4, 167.7
(2 ꢂ CO2Me), 168.8, 171.8 (2 ꢂ TZD C-4), 172.3,
General procedure for the preparation of pyraz-
ole–TZDs 8a–d from 4, 5a–d, and 7. To an ice-cooled
and stirred solution of 4 (1.17 g, 10 mmol) in tetrahy-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

308
S. Nishida, H. Maruoka, Y. Yoshimura, T. Goto, R. Tomita, E. Masumoto,
F. Okabe, K. Yamagata, and T. Fujioka
Vol 49
174.6 ppm (2 ꢂ TZD C-2); ms: m/z 775 [MþH]þ.
Anal. Calcd. for C36H34N6O10S2: C, 55.80; H, 4.42; N,
10.85. Found: C, 55.57; H, 4.67; N, 10.67.
Anal. Calcd. for C40H42N6O10S2: C, 57.82; H, 5.09; N,
10.11. Found: C, 57.42; H, 5.16; N, 10.13.
10,40,60,70-Tetrahydro-60-(1,2-dihydro-1,5-dimethyl-3-
oxo-2-phenyl-3H-pyrazol-4-yl)-10-methyl-40-[(2,4-dioxo-
3-thiazolidine)acetic acid tert-butyl ester-5-yl]spiro[-
thiazolidine-5,50-[5H]indazol]-2,30,4(20H)-trione-3-acetic
acid tert-butyl ester (9d).. This compound was obtained
as colorless needles, mp 155–157ꢁC dec. (acetone–
petroleum ether); IR (potassium bromide): m 1742, 1694
cmꢀ1 (C¼¼O); 1H NMR (deuteriochloroform): d 1.27–
1.47 (m, 18H, 2 ꢂ CO2CMe3), 2.20 (s, 3H, pyrazole
5-Me), 2.87–2.95 (m, 1H, 70-H), 3.03, 3.20 (s, 6H, 2 ꢂ
pyrazole 1-Me), 3.79–3.84 (m, 1H, 60-H), 4.21, 4.26
(s, 4H, 2 ꢂ NCH2), 4.23–4.25 (m, 2H, 40- and 70-H),
4.85 (s, 1H, TZD 5-H), 7.24–7.28 (m, 3H, Ph-H),
7.32–7.48 ppm (m, 7H, Ph-H); 13C NMR (deuterio-
chloroform): d 11.6 (pyrazole 5-Me), 23.7 (C-70), 27.9,
27.95, 27.99, 28.0 (2 ꢂ CO2CMe3), 35.0, 35.6 (2 ꢂ
pyrazole 1-Me), 39.6 (C-60), 42.9, 43.2 (2 ꢂ NCH2),
43.9 (TZD C-5), 49.8 (C-40), 69.8 (C-50), 82.4, 83.1 (2
ꢂ CO2CMe3), 101.3 (pyrazole C-4), 102.5 (C-30a),
124.5, 124.9, 127.0, 127.2, 129.18, 129.2, 134.5, (Ph-
C), 154.5 (pyrazole C-5 and C-70a), 163.3 (C-30),
164.6 (pyrazole C-3), 165.1, 166.3 (2 ꢂ CO2But),
168.7, 171.8 (2 ꢂ TZD C-4), 172.3, 174.6 ppm (2 ꢂ
TZD C-2); ms: m/z 859 [MþH]þ. Anal. Calcd. for
C42H46N6O10S2: C, 58.73; H, 5.40; N, 9.78. Found: C,
59.01; H, 5.51; N, 9.81.
10,40,60,70-Tetrahydro-60-(1,2-dihydro-1,5-dimethyl-
3-oxo-2-phenyl-3H-pyrazol-4-yl)-10-methyl-40-[(2,4-dioxo-
3-thiazolidine)acetic acid ethyl ester-5-yl]spiro[thiazo-
lidine-5,50-[5H]indazol]-2,30,4(20H)-trione-3-acetic acid
ethyl ester (9b). This compound was obtained as color-
less needles, mp 148–150ꢁC dec. (acetone–petroleum
ether); IR (potassium bromide): m 1746, 1693 cmꢀ1
1
(C¼¼O); H NMR (deuteriochloroform): d 1.23–1.30 (m,
6H, 2 ꢂ CO2CH2Me), 2.18 (s, 3H, pyrazole 5-Me), 2.59–
2.64 (m, 1H, 70-H), 3.01, 3.14 (s, 6H, 2 ꢂ pyrazole 1-
Me), 3.80–3.83 (m, 1H, 60-H), 4.13–4.24 (m, 4H, 2 ꢂ
CO2CH2Me), 4.32–4.41 (m, 2H, 40- and 70-H), 4.32, 4.40
(s, 4H, 2 ꢂ NCH2), 4.85 (s, 1H, TZD 5-H), 7.25–7.34
(m, 6H, Ph-H), 7.38–7.50 ppm (m, 4H, Ph-H); 13C NMR
(deuteriochloroform): d 11.0 (pyrazole 5-Me), 14.0, 14.1
(2 ꢂ CO2CH2Me), 23.3 (C-70), 35.3, 35.5 (2 ꢂ pyrazole
1-Me), 39.7 (C-60), 42.2, 42.5 (2 ꢂ NCH2), 43.9 (TZD
C-5), 49.7 (C-40), 61.8, 62.1 (2 ꢂ CO2CH2Me), 69.8 (C-
50), 100.9 (pyrazole C-4), 102.5 (C-30a), 124.5, 124.9,
126.1, 127.0, 129.15, 129.2, 134.3, 134.5, (Ph-C), 154.4
(C-70a), 154.8 (pyrazole C-5), 163.3 (C-30), 164.8 (pyraz-
ole C-3), 165.9, 167.3 (2 ꢂ CO2Et), 168.9, 171.9 (2 ꢂ
TZD C-4), 172.4, 174.6 ppm (2 ꢂ TZD C-2); ms: m/z
803 [MþH]þ; high-resolution ms: Calcd. for
C38H39N6O10S2 803.2169, found 803.2166. Anal. Calcd.
for C38H38N6O10S2ꢃ0.8H2O: C, 55.84; H, 4.88; N, 10.28.
Found: C, 55.75; H, 4.92; N, 10.15.
Tumor cell culture and MTT assay.. A549 and
HeLa cells were obtained from DS Pharma Biomedical
(Osaka, Japan). B16F10 cells were purchased from
American Type Culture Collection (Manassas, VA).
Tumor cells were cultured in Roswell Park Memorial
Institute 1640 medium or Dulbecco’s Modified Eagle
Medium (DMEM) at 37ꢁC with 5% carbon dioxide and
95% air, supplemented with fetal bovine serum and/or
penicillin, streptomycin, or kanamycin. The cells were
seeded onto 96-well plates. The inhibition of the cellular
growth was estimated using MTT assay according to
Mosmann [41] and our previous methods [42].
10,40,60,70-Tetrahydro-60-(1,2-dihydro-1,5-dimethyl-3-
oxo-2-phenyl-3H-pyrazol-4-yl)-10-methyl-40-[(2,4-dioxo-
3-thiazolidine)acetic acid isopropyl ester-5-yl]spiro[-
thiazolidine-5,50-[5H]indazol]-2,30,4(20H)-trione-3-
acetic acid isopropyl ester (9c).. This compound was
obtained as colorless needles, mp 170–172ꢁC dec. (ace-
tone–petroleum ether); IR (potassium bromide): m 1741,
1695 cmꢀ1 (C¼¼O); 1H NMR (deuteriochloroform): d
1.19–1.27 (m, 12H, 2 ꢂ CO2CHMe2), 2.17 (s, 3H, pyr-
azole 5-Me), 2.60–2.64 (m, 1H, 70-H), 3.01, 3.14 (s, 6H,
2 ꢂ pyrazole 1-Me), 3.78–3.83 (m, 1H, 60-H), 4.27,
4.36 (s, 4H, 2 ꢂ NCH2), 4.31–4.32 (m, 2H, 40- and 70-
H), 4.85 (s, 1H, TZD 5-H), 4.84–5.07 (m, 2H, 2 ꢂ
CO2CHMe2), 7.23–7.58 ppm (m, 10H, Ph-H); 13C NMR
(deuteriochloroform): d 11.6 (pyrazole 5-Me), 21.5,
21.6, 21.7, 21.8 (2 ꢂ CO2CHMe2), 23.4 (C-70), 35.3,
35.6 (2 ꢂ pyrazole 1-Me), 39.8 (C-60), 42.4, 42.8 (2 ꢂ
NCH2), 43.9 (TZD C-5), 49.7 (C-40), 69.4, 70.2 (2 ꢂ
CO2CHMe2), 69.8 (C-50), 101.1 (pyrazole C-4), 102.6
(C-30a), 124.2, 124.6, 125.0, 125.7, 126.9, 127.2, 129.1,
129.2, 134.5, 134.6, (Ph-C), 154.6 (pyrazole C-5), 154.8
(C-70a), 163.4 (C-30), 164.7 (pyrazole C-3), 165.4, 166.7
(2 ꢂ CO2Pri), 168.8, 171.2 (2 ꢂ TZD C-4), 172.4,
174.6 ppm (2 ꢂ TZD C-2); ms: m/z 831 [MþH]þ.
3T3-L1 cell culture and differentiation assay. 3T3-
L1 preadipocytes, which were obtained from DS Pharma
Biomedical (Japan), were cultured in DMEM and differ-
entiated by a modified previously described protocol
[43]. After 2 days, postconfluent preadipocytes were
treated for 2 days with complete medium containing
dexamethasone (1 lM), insulin (0.17 lM), and 3-
mehtyl-1-isobutylxanthine (0.5 mM). After medium
replacement, they were incubated for further 2 days
with postdifferentiation medium and for the last 2 days
with only complete medium. The medium was changed
every 2 days. Cells were treated the whole time period
with either dimethyl sulfoxide-d6 (DMSO) as a negative
control, pioglitazone as a positive control, or the
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

March 2012
Synthesis and Biological Activities of Some New Thiazolidine
Derivatives Containing Pyrazole Ring System
309
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synthesized compounds until day 12 of differentiation,
after which the cells were washed with phosphate-buf-
fered saline and stained with Oil Red O. For quantifica-
tion, the dye was extracted with isopropanol and the
absorption was measured at 540 nm.
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Acknowledgment. The authors thank Mr. Hiroshi Hanazono
and Ms. Yukiko Iwase for obtaining Mass spectra and NMR spec-
tra and Ms. Junko Honda for her valuable help with elemental
analyses.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet