Chemical reactivity and application of 4-alkylidene-3H-pyrazol-3-ones: Synthesis and antifungal activity of polysubstituted pyrazoles

Article abstract of DOI:10.3987/COM-15-S(T)13

Chemical reactivity and application of 4-alkylidene-3H-pyrazol-3-ones are described. Furthermore, twelve of the newly synthesized O-substituted pyrazoles were evaluated for their antifungal activity in vitro against Candida albicans and Saccharomyces cere

Full text of DOI:10.3987/COM-15-S(T)13

HETEROCYCLES, Vol. 93, No. 1, 2016, pp. -. © 2016 The Japan Institute of Heterocyclic Chemistry
Received, 17th August, 2015, Accepted, 11th, September, 2015, Published online, 29th October, 2015
DOI: 10.3987/COM-15-S(T)13
CHEMICAL REACTIVITY AND APPLICATION OF 4-ALKYLIDENE-
3H-PYRAZOL-3-ONES: SYNTHESIS AND ANTIFUNGAL ACTIVITY OF
POLYSUBSTITUTED PYRAZOLES
Hiroshi Maruoka,*,a Masataka Hokao,a Nobuhiro Kashige,a Eiichi
Masumoto,a Fumi Okabe,a Reiko Tanaka,b Fumio Miake,a Toshihiro
Fujioka,a and Kenji Yamagataa
aFaculty of Pharmaceutical Sciences, Fukuoka University, 8−19−1 Nanakuma,
Jonan−ku, Fukuoka 814−0180, Japan; E-mail: maruoka@fukuoka-u.ac.jp
bMedical Mycology Research Center, Chiba University, Chuo-ku, Chiba
260-8673, Japan
Abstract – Chemical reactivity and application of 4-alkylidene-3H-pyrazol-3-
ones are described. Furthermore, twelve of the newly synthesized O-substituted
pyrazoles were evaluated for their antifungal activity in vitro against Candida
albicans and Saccharomyces cerevisiae.
Pyrazoles are important class of heterocyclic compounds, which found a widespread use in various
applications. Its derivatives have certainly been shown to exhibit various pharmaceuticals and biological
activities.1 The pyrazole ring is a constituent of a variety of natural products such as pyrazomycin2 and
L-β-(1-pyrazolyl)alanine.3 In this context, the synthesis of pyrazole derivatives continues to attract
attention and provides an interesting challenge.4
In the course of our attempts to utilize the characteristic property of 3H-pyrazol-3-ones, we have
discussed the synthesis of 1-acyl-1,2-dihydro-3H-pyrazol-3-ones through Lewis acid-mediated re-
arrangement of 3-acyloxypyrazoles.5 More recently, as part of our study using 4-arylidene-3H-pyrazol-3-
one as the starting material for the preparation of spiro-derivatives of pyrazole, we have reported a simple
method for the synthesis of new spirocyclopropanepyrazoles 3a−d, followed by the reaction of
4-arylidene-3H-pyrazol-3-one 1 with α-chloro esters 2a−d (Scheme 1).6 The synthesis of 4-alkylidene-
and 4-arylidene-3H-pyrazol-3-ones has been the subject of numerous publications over the past several
decades.7 While a great number of compounds of this structural type have been prepared, there are
relatively few reports describing the chemical reactivity of these compounds, particularly the 4-alkylidene
derivatives.7d,7f,8 Thus, to expand the scope application of reactions of 4-alkylidene-3H-pyrazol-3-ones, we

focused our attention on the chemical reactivity of 4-alkylidene-3H-pyrazol-3-ones in the presence of a
base and now present the results of our investigation, an efficient method for preparing new
polysubstituted pyrazole derivatives with antifungal activity.
R
Ph
Me
Me
Ph
Ph
Ph
NaH
DMF
+
ClCH2R
2a−d
N
N
O
O
N
N
Ph
Ph
3a−d
1
a: R = CO2Me, b: R = CO2Et, c: R = CO2Pri, d: R = CO2But
R
Me
Me
Me
N
O
N
Me
Me
ClCH2R
2a−d
Ph
[not obtained]
Me
N
O
N
Me
Ph
Me
4
NaH
DMF
N
O
R
N
Ph
5a−d
Scheme 1
The starting materials, 4-alkylidene derivatives 4, 6, and 8, were prepared by treatment of
2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one and ketones such as acetone, cyclopentanone, and
cycloheptanone according to the method reported in literature.7 With the aim of extending the
preparation of spiro-derivatives of pyrazole, we examined the reaction of 4 with α-chloro esters 2a−d, but
the reaction mode was completely changed (Scheme 1). Indeed, when a mixture of 4-isopropylidene-3H-
pyrazol-3-one 4 with 2a−d in the presence of NaH in DMF was stirred at 80 °C for 1 h, the corresponding
O-substituted pyrazoles 5a−d were isolated in 44, 37, 27, and 32% yields, respectively. In this case, the
reaction was not clean and the expected spiro-compounds could not be detected at all.
The skeleton of 4 probably exists in three tautomeric forms as shown in Figure 1. However, our
observation indicates that in the presence of a base, a deprotonation of 4 easily occurs and then
O-substituted pyrazoles 5a−d would be produced from OH form via O-attack nucleophilic substitution.

The reason for this chemical reactivity of 4 is not clear at present. Thus, it seems that the straightforward
preparation of spiro-compounds9 by the reaction of 4 with 2a−d is not easy.
Me
Me
Me
Me
Me
NH
Me
HO
Me
N
N
O
O
N
N
N
Ph
Ph
CH form
Ph
OH form
NH form
Figure 1. Tautomeric forms of compound 4
Me
N
Me
K2CO3
ClCH2R
2a−d
+
7a (64%)
N
DMF
O
O
7b (63%)
7c (57%)
7d (52%)
R
N
N
Ph
Ph
6
Me
Me
K2CO3
DMF
ClCH2R
2a−d
+
9a (91%)
9b (72%)
9c (81%)
9d (65%)
N
N
O
O
R
N
N
Ph
Ph
8
a: R = CO2Me, b: R = CO2Et, c: R = CO2Pri, d: R = CO2But
Scheme 2
Subsequently, the reactions of 4-cyclopentylidene- and 4-cycloheptylidene-3H-pyrazol-3-ones 6 and 8
with 2a−d in the presence of K2CO3 gave the corresponding O-substituted pyrazoles 7a−d and 9a−d in
moderate to good yields (Scheme 2). These products 5a−d, 7a−d, and 9a−d gave satisfactory elemental
analyses and spectroscopic data consistent with their assigned structures. For examples, the IR spectra of
1
7a−d display bands in the range of 1730−1770 cm-1 due to the ester carbonyl group. The H NMR
spectra of 7a−d exhibit a two-proton singlet near δ 4.4 attributable to the methylene protons between
13
oxygen atom and carbonyl group and a one-proton singlet near δ 5.8 due to the olefin proton. The C

NMR spectra of 7a−d show a signal near δ 70 due to the methylene carbon between oxygen atom and
carbonyl group, a signal near δ 104 due to the pyrazole C-4 carbon, a signal near δ 128 due to the
cyclopentene C-2 carbon, a signal near δ 133 due to the cyclopentene C-1 carbon, and a signal near δ 167
due to the ester carbonyl carbon (see experimental section).
H
H
Me
Me
Me
mCPBA
O
HO
R
H
N
N
THF
N
O
O
O
N
N
R
N
R
Ph
Ph
Ph
B
A
7a−d
mCPBA
OH
H
O
Me
Me
Me
O
HO
O
H
H
N
N
N
O
O
O
R
N
N
N
R
R
Ph
Ph
Ph
D
10a−d
C
Me
mCPBA
mCPBA
HO
EtO2C
7b
10b
N
CHCl3
THF
O
N
Ph
11
a: R = CO2Me, b: R = CO2Et, c: R = CO2Pri, d: R = CO2But
Scheme 3
During our study of the synthesis of polysubstituted pyrazoles, we found that O-substituted pyrazoles
7a−d were reacted with m-chloroperbenzoic acid (mCPBA) to afford the corresponding 4-(2-cyclopenten-
1-one)-substituted pyrazoles 10a−d. After some optimization, the best results were obtained when 7a−d
were treated with an excess amount of mCPBA in THF, cyclopentenone-containing pyrazoles 10a−d
were isolated in 73, 79, 92, and 89% yields (Scheme 3). The IR spectra of 10a−d display a carbonyl band
of cyclopentenone at 1706 or 1708 cm-1. The 13C NMR spectra of 10a−d show a signal at δ 207.8 due to

the carbonyl carbon of cyclopentenone. By comparison of NMR spectra, MS spectra, and elemental
analyses of 10a−d it seems that the structural assignments given to these compounds are correct.
A plausible reaction mechanism for the formation of 10a−d is illustrated in Scheme 3. The reaction of
7a−d with mCPBA probably causes an epoxidation to give the epoxides A, which would undergo ring
opening via a protonation to afford the intermediate hydroxy cyclic alkenes B. An epoxidation of B again
produces the epoxides C, which would undergo a protonation/ring-opening reaction to provide the
dihydroxy cyclic alkenes D. A dehydration of D easily occurs and then 4-(2-cyclopenten-1-one)-
substituted pyrazoles 10a−d would be formed. To our knowledge, this type of tandem epoxidation
reaction between a cyclic alkene and peroxides has not been described previously. Fortunately, we found
the reaction condition under which the key intermediate cyclopentenol-containing pyrazole 11 could be
isolated. Thus, when 7b was treated with 1.0 equivalent of mCPBA in CHCl3 at 0−5 °C overnight, 11 was
obtained in 93% yield. Treatment of compound 11 with 1.0 equivalent of mCPBA in THF produced 10b
(84%), which was identical with an authentic sample prepared from 7b and an excess amount of mCPBA
in THF according to Scheme 3. Therefore, it appears that 4-(2-cyclopenten-1-one)-substituted pyrazoles
10a−d may be formed directly from O-substituted pyrazoles 7a−d, perhaps by a mechanism involving
epoxidation/ring-opening to cyclopentenol-containing pyrazoles, followed by an epoxidation/dehydration
sequence.
R1 R2
Me
Me
K2CO3
ClCH(R1)R2
+
N
N
DMF
O
O
N
N
Ph
a: R1 = Me, R2 = CN
b: R1 = H, R2 = COPh
Ph
12a (63%)
12b (91%)
6
Scheme 4
In addition to the reactions described above, the behavior of the other reagents was also investigated.
Interestingly, when a mixture of compound 6 with 2-chloropropionitrile or 2-chloroacetophenone in the
presence of K2CO3 in DMF was stirred at 80 °C for 1 h or room temperature overnight, the corresponding
C-substituted pyrazoles 12a,b were isolated in 63 and 91% yields, respectively (Scheme 4). In this case,
O-substituted pyrazoles such as 7a−d could not be obtained at all. The reason for this change of behavior
is not clear at present.

Table 1. In vitro antifungal activity of 5a−d, 7a−d, and 9a−d against C. albicans and S. cerevisiae.
MIC (µg/mL)
Entry
Compound
C. albicansa
13c
S. cerevisiaeb
1
2
5a
13c
13c
5b
13c
3
5c
5d
13c
13c
4
13c
13c
5
7a
7b
13c
13c
6
13c
13c
7
7c
7d
13c
13c
8
13c
13c
9
9a
9b
13c
13c
10
11
12
13
14
15
16
13c
13c
9c
13c
13c
9d
13c
13c
Miconazole
Itraconazole
DMSO
EtOH
2d
0.5e
4f
2d
> 12.5%
> 12.5%
> 6.25%
> 6.25%
a RPMI1640 medium, 37 °C. b YM medium, 25 °C. c containing 1.0% DMSO.
d containing 0.015% DMSO. e containing 0.04% DMSO. f containing 0.03% DMSO.
Twelve of the newly synthesized O-substituted pyrazole derivatives 5a−d, 7a−d, and 9a−d were tested
for their antifungal activity in vitro against Candida albicans and Saccharomyces cerevisiae. In vitro
susceptibility tests were performed to evaluate minimum inhibitory concentrations (MICs) using the
method described in the guidelines of NCCLS Document M27-A210 and our previous paper.6 Miconazole
and Itraconazole were used as standard drugs for comparison of the antifungal activity. The results
obtained are summarized in Table 1. All the synthesized compounds were active against both Candida
and Saccharomyces with MIC ≥ 13 µg/mL (entries 1−12). It is worth noting that the entire tested
compounds 5a−d, 7a−d, and 9a−d showed more potent antifungal activity than those of the
spirocyclopropanepyrazoles 3a−d, which were active against Candida with MIC ≥ 25 µg/mL and
Saccharomyces with MIC ≥ 50 µg/mL.6
In conclusion, we have demonstrated the reactions of 4-alkylidene-3H-pyrazol-3-ones 4, 6, and 8 with
α-chloro esters in the presence of a base. This methodology offers significant advantages with regard to
the supply of polysubstituted pyrazole derivatives, which may be important building blocks in organic
synthesis and for the preparation of biologically active compounds with interest in medicinal chemistry.
In this present work, we showed that polysubstituted pyrazoles 5a−d, 7a−d, and 9a−d were moderately to
fairly active against the tested fungi whereas the reference drug such as Miconazole and Itraconazole
exhibited good to excellent results against both the fungi. Our results suggest that 5a−d, 7a−d, and 9a−d

would play a role in vivo. At the present time, we continue to investigate the applications of
4-alkylidene-3H-pyrazol-3-ones.
EXPERIMENTAL
The IR spectra were recorded on a JASCO FT/IR-4100 spectrometer. The 1H and 13C NMR spectra were
1
13
measured with a JEOL JNM-A500 spectrometer at 500 and 125 MHz, respectively. The H and C
chemical shifts (δ) are reported in parts per million (ppm) relative to TMS as internal standard. Positive
FAB MS spectra were obtained on a JEOL JMS-700T spectrometer. Elemental analyses were performed
on YANACO MT-6 CHN analyzer. The starting compounds, 4-alkylidene-3H-pyrazol-3-ones 4, 6, and 8,
were prepared in this laboratory according to the procedure reported in literature.7
General procedure for the preparation of O-substituted pyrazoles 5a−d from 4-isopropylidene-3H-
pyrazol-3-one 4 and α-chloro esters 2a−d. To an ice-cooled and stirred solution of 4 (0.428 g, 2 mmol)
in DMF (10 mL), 60% NaH (0.160 g, 4 mmol) was added. The stirring was continued at rt until evolution
of gas ceased. To the obtained solution, methyl chloroacetate (2a) (0.651 g, 6 mmol), ethyl chloroacetate
(2b) (0.735 g, 6 mmol), isopropyl chloroacetate (2c) (0.819 g, 6 mmol), or tert-butyl chloroacetate (2d)
(0.904 g, 6 mmol) was added with stirring and ice-cooling, and then the mixture was stirred at 80 ˚C for 1
h. After removal of the solvent in vacuo, cold H2O was added to the residue. The resulting mixture was
extracted with CHCl3 (60 mL). The extract was dried over anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography on silica gel with CHCl3 as the eluent to give 5a−d.
Methyl {[3-methyl-1-phenyl-4-(propen-2-yl)-1H-pyrazol-5-yl]oxy}acetate (5a): Pale yellow oil (0.252
1
g, 44%); IR (neat): ν 1765, 1745 cm-1 (C=O); H NMR (CDCl3): δ 2.07 [dd, J = 0.9, 1.5 Hz, 3H,
CH2=C(Me)-], 2.27 (s, 3H, pyrazole 3-Me), 3.67 (s, 3H, OMe), 4.54 (s, 2H, OCH2), 5.04−5.05 (m, 1H,
olefin H), 5.20−5.21 (m, 1H, olefin H), 7.26−7.28 (m, 1H, Ph-H), 7.40−7.43 (m, 2H, Ph-H), 7.70−7.72 (m,
2H, Ph-H); 13C NMR (CDCl3): δ 13.9 (pyrazole 3-Me), 23.5 [CH2=C(Me)-], 52.1 (CO2Me), 69.2 (OCH2),
108.2 (pyrazole C-4), 116.2 (CH2=), 122.5, 126.5, 128.9 (Ph-C), 135.6 [CH2=C(Me)-], 138.5 (Ph-C),
146.9 (pyrazole C-3), 148.9 (pyrazole C-5), 168.1 (C=O); MS: m/z 287 [M+H]+. Anal. Calcd for
C16H18N2O3: C, 67.12; H, 6.34; N, 9.78. Found: C, 67.01; H, 6.36; N, 9.76.
Ethyl {[3-methyl-1-phenyl-4-(propen-2-yl)-1H-pyrazol-5-yl]oxy}acetate (5b): Pale yellow oil (0.222 g,
37%); IR (neat): ν 1762, 1739 cm-1 (C=O); 1H NMR (CDCl3): δ 1.20 (t, J = 7.0 Hz, 3H, CO2CH2Me), 2.08
[dd, J = 0.9, 1.5 Hz, 3H, CH2=C(Me)-], 2.27 (s, 3H, pyrazole 3-Me), 4.15 (q, J = 7.0 Hz, 2H, CO2CH2Me),
4.54 (s, 2H, OCH2), 5.04−5.06 (m, 1H, olefin H), 5.21−5.22 (m, 1H, olefin H), 7.26−7.28 (m, 1H, Ph-H),
13
7.40−7.43 (m, 2H, Ph-H), 7.72−7.74 (m, 2H, Ph-H); C NMR (CDCl3): δ 13.9 (pyrazole 3-Me), 14.0
(CO2CH2Me), 23.6 [CH2=C(Me)-], 61.4 (CO2CH2Me), 69.3 (OCH2), 108.1 (pyrazole C-4), 116.4 (CH2=),

122.5, 126.5, 128.9 (Ph-C), 135.6 [CH2=C(Me)-], 138.5 (Ph-C), 146.9 (pyrazole C-3), 149.0 (pyrazole
C-5), 167.6 (C=O); MS: m/z 301 [M+H]+. Anal. Calcd for C17H20N2O3: C, 67.98; H, 6.71; N, 9.33. Found:
C, 67.78; H, 6.71; N, 9.31.
Isopropyl {[3-methyl-1-phenyl-4-(propen-2-yl)-1H-pyrazol-5-yl]oxy}acetate (5c): Pale yellow oil
1
(0.170 g, 27%); IR (neat): ν 1757, 1735 cm-1 (C=O); H NMR (CDCl3): δ 1.18 (d, J = 6.1 Hz, 6H,
CO2CHMe2), 2.07 [dd, J = 0.9, 1.5 Hz, 3H, CH2=C(Me)-], 2.27 (s, 3H, pyrazole 3-Me), 4.53 (s, 2H,
OCH2), 5.02−5.07 (m, 2H, olefin H and CO2CHMe2), 5.21−5.23 (m, 1H, olefin H), 7.25−7.28 (m, 1H,
13
Ph-H), 7.39−7.42 (m, 2H, Ph-H), 7.73−7.75 (m, 2H, Ph-H); C NMR (CDCl3): δ 13.8 (pyrazole 3-Me),
21.6 (CO2CHMe2), 23.6 [CH2=C(Me)-], 69.2 (CO2CHMe2), 69.4 (OCH2), 108.0 (pyrazole C-4), 116.5
(CH2=), 122.4, 126.4, 128.9 (Ph-C), 135.6 [CH2=C(Me)-], 138.5 (Ph-C), 146.9 (pyrazole C-3), 149.0
(pyrazole C-5), 167.2 (C=O); MS: m/z 315 [M+H]+. Anal. Calcd for C18H22N2O3: C, 68.77; H, 7.05; N,
8.91. Found: C, 68.64; H, 7.12; N, 8.93.
tert-Butyl {[3-methyl-1-phenyl-4-(propen-2-yl)-1H-pyrazol-5-yl]oxy}acetate (5d): Pale yellow oil
(0.210 g, 32%); IR (neat): ν 1756, 1731 cm-1 (C=O); 1H NMR (CDCl3): δ 1.41 (s, 9H, CO2CMe3), 2.07 (m,
3H, CH2=C(Me)-), 2.27 (s, 3H, pyrazole 3-Me), 4.45 (s, 2H, OCH2), 5.04−5.05 (m, 1H, olefin H),
5.22−5.23 (m, 1H, olefin H), 7.25−7.27 (m, 1H, Ph-H), 7.39−7.42 (m, 2H, Ph-H), 7.74−7.76 (m, 2H,
Ph-H); 13C NMR (CDCl3): δ 13.8 (pyrazole 3-Me), 23.7 [CH2=C(Me)-], 27.8, 28.0, 28.1 (CO2CMe3), 69.7
(OCH2), 82.5 (CO2CMe3), 107.9 (pyrazole C-4), 116.5 (CH2=), 122.4, 126.4, 128.9 (Ph-C), 135.7
[CH2=C(Me)-], 138.5 (Ph-C), 146.9 (pyrazole C-3), 149.1 (pyrazole C-5), 166.7 (C=O); MS: m/z 329
[M+H]+. Anal. Calcd for C19H24N2O3: C, 69.49; H, 7.37; N, 8.53. Found: C, 69.49; H, 7.31; N, 8.59.
General procedure for the preparation of O-substituted pyrazoles 7a−d and 9a−d from
4-cyclopentylidene- or 4-cycloheptylidene-3H-pyrazol-3-one 6 or 8 and α-chloro esters 2a−d. A
mixture of 6 (0.480 g, 2 mmol) or 8 (0.536 g, 2 mmol), methyl chloroacetate (2a) (0.651 g, 6 mmol),
ethyl chloroacetate (2b) (0.735 g, 6 mmol), isopropyl chloroacetate (2c) (0.819 g, 6 mmol), or tert-butyl
chloroacetate (2d) (0.904 g, 6 mmol), and K2CO3 (0.553 g, 4 mmol) in DMF (10 mL) was stirred at rt
overnight. After removal of the solvent in vacuo, cold H2O was added to the residue. The resulting
mixture was extracted with CHCl3 (60 mL). The extract was dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by column chromatography on silica gel with CHCl3 as
the eluent to afford 7a−d and 9a−d.
Methyl {[4-(cyclopenten-1-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (7a): Pale yellow oil
(0.401 g, 64%); IR (neat): ν 1766, 1745 cm-1 (C=O); 1H NMR (CDCl3): δ 1.94−1.99 (m, 2H, cyclopentene
4-H), 2.31 (s, 3H, pyrazole 3-Me), 2.46−2.51 (m, 2H, cyclopentene 3- or 5-H), 2.65−2.69 (m, 2H,
cyclopentene 3- or 5-H), 3.68 (s, 3H, OMe), 4.44 (s, 2H, OCH2), 5.70−5.86 (m, 1H, olefin H), 7.25−7.29

(m, 1H, Ph-H), 7.37−7.43 (m, 2H, Ph-H), 7.69−7.71 (m, 2H, Ph-H); 13C NMR (CDCl3): δ 14.6 (pyrazole
3-Me), 23.4 (cyclopentene C-4), 32.9, 35.3 (cyclopentene C-3 and -5), 52.1 (CO2Me), 69.5 (OCH2), 104.4
(pyrazole C-4), 122.4, 126.6 (Ph-C), 128.2 (cyclopentene C-2), 129.0 (Ph-C), 133.3 (cyclopentene C-1),
138.4 (Ph-C), 147.4 (pyrazole C-3), 149.1 (pyrazole C-5), 167.9 (C=O); MS: m/z 313 [M+H]+. Anal.
Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97. Found: C, 69.09; H, 6.53; N, 8.93.
Ethyl {[4-(cyclopenten-1-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (7b): Pale yellow oil
1
(0.410 g, 63%); IR (neat): ν 1762, 1739 cm-1 (C=O); H NMR (CDCl3): δ 1.20 (t, J = 7.0 Hz, 3H,
CO2CH2Me), 1.95−2.00 (m, 2H, cyclopentene 4-H), 2.31 (s, 3H, pyrazole 3-Me), 2.47−2.50 (m, 2H,
cyclopentene 3- or 5-H), 2.66−2.69 (m, 2H, cyclopentene 3- or 5-H), 4.16 (q, J = 7.0 Hz, 2H,
CO2CH2Me), 4.43 (s, 2H, OCH2), 5.84−5.85 (m, 1H, olefin H), 7.25−7.28 (m, 1H, Ph-H), 7.40−7.43 (m,
2H, Ph-H), 7.71−7.72 (m, 2H, Ph-H); 13C NMR (CDCl3): δ 14.0 (CO2CH2Me), 14.6 (pyrazole 3-Me), 23.4
(cyclopentene C-4), 32.9, 35.4 (cyclopentene C-3 and -5), 61.3 (CO2CH2Me), 69.6 (OCH2), 104.3
(pyrazole C-4), 122.4, 126.5 (Ph-C), 128.2 (cyclopentene C-2), 129.0 (Ph-C), 133.3 (cyclopentene C-1),
138.4 (Ph-C), 147.4 (pyrazole C-3), 149.2 (pyrazole C-5), 167.5 (C=O); MS: m/z 327 [M+H]+. Anal.
Calcd for C19H22N2O3: C, 69.92; H, 6.79; N, 8.58. Found: C, 69.74; H, 6.85; N, 8.54.
Isopropyl {[4-(cyclopenten-1-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (7c): Pale yellow oil
1
(0.388 g, 57%); IR (neat): ν 1757, 1734 cm-1 (C=O); H NMR (CDCl3): δ 1.19 (d, J = 6.1 Hz, 6H,
CO2CHMe2), 1.94−2.04 (m, 2H, cyclopentene 4-H), 2.30 (s, 3H, pyrazole 3-Me), 2.47−2.50 (m, 2H,
cyclopentene 3- or 5-H), 2.65−2.69 (m, 2H, cyclopentene 3- or 5-H), 4.41 (s, 2H, OCH2), 5.05 (sep, J =
6.1 Hz, 1H, CO2CHMe2), 5.84−5.85 (m, 1H, olefin H), 7.24−7.28 (m, 1H, Ph-H), 7.39−7.42 (m, 2H,
13
Ph-H), 7.71−7.74 (m, 2H, Ph-H); C NMR (CDCl3): δ 14.6 (pyrazole 3-Me), 21.6 (CO2CHMe2), 23.4
(cyclopentene C-4), 32.9, 35.4 (cyclopentene C-3 and -5), 69.2 (CO2CHMe2), 69.8 (OCH2), 104.3
(pyrazole C-4), 122.3, 126.5 (Ph-C), 128.2 (cyclopentene C-2), 129.0 (Ph-C), 133.4 (cyclopentene C-1),
138.5 (Ph-C), 147.4 (pyrazole C-3), 149.3 (pyrazole C-5), 167.1 (C=O); MS: m/z 341 [M+H]+. Anal.
Calcd for C20H24N2O3: C, 70.56; H, 7.11; N, 8.23. Found: C, 70.46; H, 7.08; N, 8.24.
tert-Butyl {[4-(cyclopenten-1-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (7d): Pale yellow oil
1
(0.368 g, 52%); IR (neat): ν 1757, 1731 cm-1 (C=O); H NMR (CDCl3): δ 1.42 (s, 9H, CO2CMe3),
1.94−1.99 (m, 2H, cyclopentene 4-H), 2.31 (s, 3H, pyrazole 3-Me), 2.47−2.51 (m, 2H, cyclopentene 3- or
5-H), 2.66−2.69 (m, 2H, cyclopentene 3- or 5-H), 4.33 (s, 2H, OCH2), 5.85−5.86 (m, 1H, olefin H),
7.24−7.28 (m, 1H, Ph-H), 7.39−7.42 (m, 2H, Ph-H), 7.72−7.74 (m, 2H, Ph-H); 13C NMR (CDCl3): δ 14.6
(pyrazole 3-Me), 23.4 (cyclopentene C-4), 28.0 (CO2CMe3), 32.9, 35.4 (cyclopentene C-3 and -5), 70.1
(OCH2), 82.5 (CO2CMe3), 104.2 (pyrazole C-4), 122.3, 126.5 (Ph-C), 128.2 (cyclopentene C-2), 129.0
(Ph-C), 133.4 (cyclopentene C-1), 138.4 (Ph-C), 147.4 (pyrazole C-3), 149.3 (pyrazole C-5), 166.5

(C=O); MS: m/z 355 [M+H]+. Anal. Calcd for C21H26N2O3: C, 71.16; H, 7.39; N, 7.90. Found: C, 71.03; H,
7.44; N, 7.90.
Methyl {[4-(cyclohepten-1-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (9a): Pale yellow oil
(0.619 g, 91%); IR (neat): ν 1765, 1745 cm-1 (C=O); 1H NMR (CDCl3): δ 1.58−1.65 (m, 4H, cycloheptene
2CH2), 1.80−1.84 (m, 2H, cycloheptene CH2), 2.22 (s, 3H, pyrazole 3-Me), 2.24−2.28 (m, 2H,
cycloheptene CH2), 2.43−2.45 (m, 2H, cycloheptene CH2), 3.67 (s, 3H, OMe), 4.62 (s, 2H, OCH2), 5.91 (t,
J = 6.7 Hz, 1H, olefin H), 7.22−7.26 (m, 1H, Ph-H), 7.38−7.41 (m, 2H, Ph-H), 7.70−7.72 (m, 2H, Ph-H);
13C NMR (CDCl3): δ 13.6 (pyrazole 3-Me), 26.8, 26.9, 28.9, 32.3, 34.3 (cycloheptene 5CH2), 52.0
(CO2Me), 68.9 (OCH2), 110.1 (pyrazole C-4), 122.4, 126.1, 128.7 (Ph-C), 134.0 (cycloheptene C-2),
134.8 (cycloheptene C-1), 138.7 (Ph-C), 147.1 (pyrazole C-3), 148.2 (pyrazole C-5), 168.2 (C=O); MS:
m/z 341 [M+H]+. Anal. Calcd for C20H24N2O3 0.3H2O: C, 69.46; H, 7.00; N, 8.10. Found: C, 69.45; H,
7.05; N, 8.10.
Ethyl {[4-(cyclohepten-1-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (9b): Pale yellow oil
1
(0.510 g, 72%); IR (neat): ν 1763, 1739 cm-1 (C=O); H NMR (CDCl3): δ 1.19 (t, J = 7.0 Hz, 3H,
CO2CH2Me), 1.57−1.66 (m, 4H, cycloheptene 2CH2), 1.80−1.84 (m, 2H, cycloheptene CH2), 2.22 (s, 3H,
pyrazole 3-Me), 2.22−2.28 (m, 2H, cycloheptene CH2), 2.43−2.45 (m, 2H, cycloheptene CH2), 4.15 (q, J
= 7.0 Hz, 2H, CO2CH2Me), 4.62 (s, 2H, OCH2), 5.91 (t, J = 6.4 Hz, 1H, olefin H), 7.22−7.26 (m, 1H,
13
Ph-H), 7.38−7.41 (m, 2H, Ph-H), 7.72−7.74 (m, 2H, Ph-H); C NMR (CDCl3): δ 13.6 (pyrazole 3-Me),
14.0 (CO2CH2Me), 26.8, 27.0, 28.9, 32.4, 34.4 (cycloheptene 5CH2), 61.3 (CO2CH2Me), 69.1 (OCH2),
110.1 (pyrazole C-4), 122.4, 126.1, 128.8 (Ph-C), 134.0 (cycloheptene C-2), 134.8 (cycloheptene C-1),
138.7 (Ph-C), 147.1 (pyrazole C-3), 148.3 (pyrazole C-5), 167.8 (C=O); MS: m/z 355 [M+H]+. Anal.
Calcd for C21H26N2O3 0.3H2O: C, 70.09; H, 7.28; N, 7.78. Found: C, 70.08; H, 7.44; N, 7.71.
Isopropyl {[4-(cyclohepten-1-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (9c): Pale yellow oil
1
(0.596 g, 81%); IR (neat): ν 1758, 1735 cm-1 (C=O); H NMR (CDCl3): δ 1.17 (d, J = 6.4 Hz, 6H,
CO2CHMe2), 1.58−1.66 (m, 4H, cycloheptene 2CH2), 1.80−1.84 (m, 2H, cycloheptene CH2), 2.22 (s, 3H,
pyrazole 3-Me), 2.25−2.29 (m, 2H, cycloheptene CH2), 2.43−2.45 (m, 2H, cycloheptene CH2), 4.61 (s, 2H,
OCH2), 5.05 (sep, J = 6.4 Hz, 1H, CO2CHMe2), 5.91 (t, J = 6.4 Hz, 1H, olefin H), 7.22−7.26 (m, 1H,
13
Ph-H), 7.38−7.41 (m, 2H, Ph-H), 7.74−7.76 (m, 2H, Ph-H); C NMR (CDCl3): δ 13.6 (pyrazole 3-Me),
21.6 (CO2CHMe2), 26.8, 27.0, 28.9, 32.4, 34.5 (cycloheptene 5CH2), 69.1 (OCH2 and CO2CHMe2), 110.0
(pyrazole C-4), 122.3, 126.0, 128.7 (Ph-C), 134.0 (cycloheptene C-2), 134.9 (cycloheptene C-1), 138.7
(Ph-C), 147.1 (pyrazole C-3), 148.3 (pyrazole C-5), 167.4 (C=O); MS: m/z 369 [M+H]+. Anal. Calcd for
C22H28N2O3 0.2H2O: C, 71.02; H, 7.59; N, 7.53. Found: C, 71.02; H, 7.72; N, 7.49.

tert-Butyl {[4-(cyclohepten-1-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (9d): Pale yellow oil
1
(0.500 g, 65%); IR (neat): ν 1756, 1732 cm-1 (C=O); H NMR (CDCl3): δ 1.41 (s, 9H, CO2CMe3),
1.58−1.66 (m, 4H, cycloheptene 2CH2), 1.81−1.85 (m, 2H, cycloheptene CH2), 2.22 (s, 3H, pyrazole
3-Me), 2.25−2.29 (m, 2H, cycloheptene CH2), 2.43−2.45 (m, 2H, cycloheptene CH2), 4.54 (s, 2H, OCH2),
5.91 (t, J = 6.4 Hz, 1H, olefin H), 7.21−7.26 (m, 1H, Ph-H), 7.37−7.40 (m, 2H, Ph-H), 7.74−7.76 (m, 2H,
13
Ph-H); C NMR (CDCl3): δ 13.6 (pyrazole 3-Me), 26.8, 27.0 (cycloheptene 2CH2), 28.0 (CO2CMe3),
28.9, 32.4, 34.6 (cycloheptene 3CH2), 69.3 (OCH2), 82.4 (CO2CMe3), 109.9 (pyrazole C-4), 122.2, 126.0,
128.7 (Ph-C), 134.0 (cycloheptene C-2), 134.9 (cycloheptene C-1), 138.8 (Ph-C), 147.1 (pyrazole C-3),
148.3 (pyrazole C-5), 166.9 (C=O); MS: m/z 383 [M+H]+. Anal. Calcd for C23H30N2O3 0.2H2O: C, 71.55;
H, 7.83; N, 7.26. Found: C, 71.55; H, 7.92; N, 7.22.
General procedure for the preparation of 4-(2-cyclopenten-1-one)-substituted pyrazoles 10a−d
from 7a−d and m-chloroperbenzoic acid. A mixture of 7a−d (1 mmol) and mCPBA (0.345 g, 2 mmol)
in THF (10 mL) was stirred at rt overnight. To the reaction mixture, a saturated aqueous NaHCO3
solution (30 mL) was added with stirring and ice-cooling. The resulting mixture was extracted with
AcOEt (60 mL). The extract was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel with CHCl3 as the eluent to provide 10a−d.
Methyl {[4-(2-cyclopenten-1-on-2-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (10a): Pale
1
yellow oil (0.239 g, 73%); IR (neat): ν 1763, 1743, 1706 cm-1 (C=O); H NMR (CDCl3): δ 2.20 (s, 3H,
pyrazole 3-Me), 2.54−2.56 (m, 2H, cyclopentenone 5-H), 2.76−2.79 (m, 2H, cyclopentenone 4-H), 3.67 (s,
3H, OMe), 4.46 (s, 2H, OCH2), 7.29−7.30 (m, 1H, Ph-H), 7.40−7.44 (m, 2H, Ph-H), 7.67 (s, 1H, olefin
13
H), 7.67−7.71 (m, 2H, Ph-H); C NMR (CDCl3): δ 13.6 (pyrazole 3-Me), 27.2 (cyclopentenone C-4),
34.4 (cyclopentenone C-5), 52.0 (CO2Me), 69.4 (OCH2), 97.9 (pyrazole C-4), 122.6, 126.9, 129.0 (Ph-C),
137.2 (cyclopentenone C-2), 138.3 (Ph-C), 147.8 (pyrazole C-3), 150.2 (pyrazole C-5), 161.6
(cyclopentenone C-3), 168.0, 207.8 (2C=O); MS: m/z 327 [M+H]+. Anal. Calcd for C18H18N2O4: C,
66.25; H, 5.56; N, 8.58. Found: C, 66.21; H, 5.64; N, 8.32.
Ethyl {[4-(2-cyclopenten-1-on-2-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (10b): Pale
yellow oil (0.269 g, 79%); IR (neat): ν 1759, 1739, 1706 cm-1 (C=O); 1H NMR (CDCl3): δ 1.20 (t, J = 7.0
Hz, 3H, CO2CH2Me), 2.20 (s, 3H, pyrazole 3-Me), 2.54−2.56 (m, 2H, cyclopentenone 5-H), 2.77−2.79 (m,
2H, cyclopentenone 4-H), 4.14 (q, J = 7.0 Hz, 2H, CO2CH2Me), 4.45 (s, 2H, OCH2), 7.26−7.30 (m, 1H,
13
Ph-H), 7.40−7.43 (m, 2H, Ph-H), 7.67 (s, 1H, olefin H), 7.66−7.73 (m, 2H, Ph-H); C NMR (CDCl3): δ
13.6 (pyrazole 3-Me), 14.0 (CO2CH2Me), 27.2 (cyclopentenone C-4), 34.4 (cyclopentenone C-5), 61.3
(CO2CH2Me), 69.6 (OCH2), 97.8 (pyrazole C-4), 122.6, 126.8, 129.0 (Ph-C), 137.4 (cyclopentenone C-2),
138.4 (Ph-C), 147.8 (pyrazole C-3), 150.3 (pyrazole C-5), 161.6 (cyclopentenone C-3), 167.6, 207.8

(2C=O); MS: m/z 341 [M+H]+. Anal. Calcd for C19H20N2O4 0.2H2O: C, 66.34; H, 5.86; N, 8.14. Found: C,
66.41; H, 6.06; N, 7.98.
Isopropyl {[4-(2-cyclopenten-1-on-2-yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (10c): Pale
yellow oil (0.326 g, 92%); IR (neat): ν 1753, 1736, 1708 cm-1 (C=O); 1H NMR (CDCl3): δ 1.17 (d, J = 6.4
Hz, 6H, CO2CHMe2), 2.20 (s, 3H, pyrazole 3-Me), 2.55−2.56 (m, 2H, cyclopentenone 5-H), 2.77−2.80 (m,
2H, cyclopentenone 4-H), 4.42 (s, 2H, OCH2), 5.02 (sep, J = 6.4 Hz, 1H, CO2CHMe2), 7.26−7.29 (m, 1H,
13
Ph-H), 7.40−7.43 (m, 2H, Ph-H), 7.72 (s, 1H, olefin H), 7.67−7.74 (m, 2H, Ph-H); C NMR (CDCl3): δ
13.7 (pyrazole 3-Me), 21.6 (CO2CHMe2), 27.2 (cyclopentenone C-4), 34.4 (cyclopentenone C-5), 69.2
(CO2CHMe2), 69.8 (OCH2), 97.8 (pyrazole C-4), 122.6, 126.7, 129.0 (Ph-C), 137.4 (cyclopentenone C-2),
138.4 (Ph-C), 147.8 (pyrazole C-3), 150.4 (pyrazole C-5), 161.5 (cyclopentenone C-3), 167.2, 207.8
(2C=O); MS: m/z 355 [M+H]+. Anal. Calcd for C20H22N2O4 0.6H2O: C, 65.77; H, 6.07; N, 7.67. Found: C,
65.75; H, 6.42; N, 7.53.
tert-Butyl {[4-(2-cyclopenten-1-on-2yl)-3-methyl-1-phenyl-1H-pyrazol-5-yl]oxy}acetate (10d): Pale
1
yellow oil (0.328 g, 89%); IR (neat): ν 1753, 1733, 1708 cm-1 (C=O); H NMR (CDCl3): δ 1.40 (s, 9H,
CO2CMe3), 2.20 (s, 3H, pyrazole 3-Me), 2.54−2.56 (m, 2H, cyclopentenone 5-H), 2.77−2.79 (m, 2H,
cyclopentenone 4-H), 4.34 (s, 2H, OCH2), 7.25−7.29 (m, 1H, Ph-H), 7.40−7.43 (m, 2H, Ph-H), 7.67 (t, J
13
= 3.0 Hz, 1H, olefin H), 7.72−7.74 (m, 2H, Ph-H); C NMR (CDCl3): δ 13.6 (pyrazole 3-Me), 27.2
(cyclopentenone C-4), 28.0 (CO2CMe3), 34.4 (cyclopentenone C-5), 70.0 (OCH2), 82.5 (CO2CMe3), 97.6
(pyrazole C-4), 122.5, 126.6, 129.0 (Ph-C), 137.4 (cyclopentenone C-2), 138.4 (Ph-C), 147.8 (pyrazole
C-3), 150.5 (pyrazole C-5), 161.6 (cyclopentenone C-3), 166.7, 207.8 (2C=O); MS: m/z 369 [M+H]+.
Anal. Calcd for C21H24N2O4 1H2O: C, 65.27; H, 6.28; N, 7.25. Found: C, 65.20; H, 6.43; N, 7.01.
The preparation of 11 from 7b and m-chloroperbenzoic acid. A mixture of 7b (0.326 g, 1 mmol) and
mCPBA (0.173 g, 1 mmol) in CHCl3 (10 mL) was stirred at 0−5 ˚C overnight. To the reaction mixture, a
saturated aqueous NaHCO3 solution (30 mL) was added with stirring and ice-cooling. The resulting
mixture was extracted with CHCl3 (60 mL). After the same work-up as described above for the
preparation
of
10a−d,
ethyl
{[4-(2-cyclopenten-1-ol-2-yl)-3-methyl-1-phenyl-1H-pyrazol-5-
yl]oxy}acetate (11) was obtained as pale yellow oil (0.319 g, 93%); IR (neat): ν 3386 (OH), 1759, 1740
1
cm-1 (C=O); H NMR (CDCl3): δ 1.23 (t, J = 7.0 Hz, 3H, CO2CH2Me), 1.89−1.92 (m, 1H, cyclopentenol
5-H), 2.32 (s, 3H, pyrazole 3-Me), 2.20−2.42 (m, 2H, cyclopentenol 4- and 5-H), 2.62−2.67 (m, 1H,
cyclopentenol 4-H), 3.39 (br, 1H, OH), 4.19 (q, J = 7.0 Hz, 2H, CO2CH2Me), 4.45 (AB q, J = 15.3 Hz,
2H, OCH2), 5.12−5.14 (m, 1H, cyclopentenol 1-H), 6.00−6.01 (m, 1H, olefin H), 7.28−7.31 (m, 1H,
Ph-H), 7.41−7.45 (m, 2H, Ph-H), 7.67−7.69 (m, 2H, Ph-H); 13C NMR (CDCl3): δ 14.0 (CO2CH2Me), 14.7
(pyrazole 3-Me), 30.5 (cyclopentenol C-4), 33.1 (cyclopentenol C-5), 61.5 (CO2CH2Me), 69.9 (OCH2),

77.9 (cyclopentenol C-1), 102.5 (pyrazole C-4), 122.6, 126.9, 129.2 (Ph-C), 130.9 (cyclopentenol C-3),
136.9 (cyclopentenol C-2), 138.4 (Ph-C), 148.0 (pyrazole C-3), 149.1 (pyrazole C-5), 167.8 (C=O); MS:
m/z 343 [M+H]+. Anal. Calcd for C19H22N2O4 0.4H2O: C, 65.28; H, 634; N, 8.01. Found: C, 65.32; H,
6.42; N, 7.77.
The preparation of 10b from 11 and m-chloroperbenzoic acid. A mixture of 11 (0.342 g, 1 mmol) and
mCPBA (0.173 g, 1 mmol) in THF (10 mL) was stirred at rt overnight. After the same work-up as
described above for the preparation of 10a−d, 10b was obtained in 84% yield (0.286 g).
General procedure for the preparation of C-substituted pyrazoles 12a,b from 4-cyclopentylidene-
3H-pyrazol-3-one 6. A mixture of 6 (0.480 g, 2 mmol), 2-chloropropionitrile (0.537 g, 6 mmol) or
2-chloroacetophenone (0.928 g, 6 mmol), and K2CO3 (0.553 g, 4 mmol) in DMF (5 mL) was stirred at 80
˚C for 1 h (in the case of the preparation of 12a) or rt overnight (in the case of the preparation of 12b).
After the same work-up as described above for the preparation of 7a−d and 9a−d, C-substituted pyrazoles
12a,b were obtained.
4-(Cyclopenten-1-yl)-4,5-dihydro-α,3-dimethyl-5-oxo-1-phenyl-1H-pyrazole-4-acetonitrile
(12a):
Pale yellow oil (0.370 g, 63%); IR (neat): ν 2242 (CN), 1714 cm-1 (C=O); 1H NMR (CDCl3): δ 1.21 (d, J
= 7.0 Hz, 3H, CHMeCN), 1.91−1.96 (m, 2H, cyclopentene 4-H), 2.32 (s, 3H, pyrazole 3-Me), 2.34−2.48
(m, 4H, cyclopentene 3- and 5-H), 3.44 (q, J = 7.0 Hz, 1H, CHMeCN), 5.85−5.86 (m, 1H, olefin H),
7.19−7.22 (m, 1H, Ph-H), 7.38−7.42 (m, 2H, Ph-H), 7.85−7.87 (m, 2H, Ph-H); 13C NMR (CDCl3): δ 13.8
(CHMeCN), 15.7 (pyrazole 3-Me), 22.7 (cyclopentene C-4), 27.9 (CHMeCN), 31.4, 32.7 (cyclopentene
C-3 and -5), 62.1 (pyrazole C-4), 118.8 (Ph-C), 119.8 (CN), 125.6, 129.0 (Ph-C), 131.4 (cyclopentene
C-2), 135.6 (cyclopentene C-1), 137.6 (Ph-C), 159.1 (pyrazole C-3), 170.4 (C=O); MS: m/z 294 [M+H]+.
Anal. Calcd for C18H19N3O 0.1H2O: C, 73.24; H, 6.49; N, 14.24. Found: C, 73.26; H, 6.58; N, 14.11.
4-(Cyclopenten-1-yl)-2,4-dihydro-5-methyl-4-(2-oxo-2-phenylethyl)-1-phenyl-3H-pyrazol-3-one
1
(12b): Pale yellow oil (0.652 g, 91%); IR (neat): ν 1704, 1685 cm-1 (C=O); H NMR (CDCl3): δ
1.87−1.94 (m, 2H, cyclopentene 4-H), 2.00 (s, 3H, pyrazole 3-Me), 2.27−2.31 (m, 2H, cyclopentene 3- or
5-H), 2.39−2.44 (m, 2H, cyclopentene 3- or 5-H), 3.80 (AB q, J = 18.1 Hz, 2H, CH2COPh), 5.81−5.82 (m,
1H, olefin H), 7.15−7.17 (m, 1H, Ph-H), 7.37−7.46 (m, 4H, Ph-H), 7.55−7.58 (m, 1H, Ph-H), 7.92−7.95
(m, 4H, Ph-H); 13C NMR (CDCl3): δ 14.2 (pyrazole 3-Me), 22.8 (cyclopentene C-4), 31.3, 32.5
(cyclopentene C-3 and -5), 41.1 (CH2COPh), 58.1 (pyrazole C-4), 119.2, 124.9, 128.2, 128.7, 128.8
(Ph-C), 129.1 (cyclopentene C-2), 133.6 (Ph-C), 135.9 (cyclopentene C-1), 138.0, 138.5 (Ph-C), 160.4
(pyrazole C-3), 173.7 (pyrazole C-5), 195.4 (C=O); MS: m/z 359 [M+H]+. Anal. Calcd for C23H22N2O2: C,
77.07; H, 6.19; N, 7.82. Found: C, 77.12; H, 6.29; N, 7.80.

Antifungal activity testing. In vitro susceptibility tests were performed to evaluate minimum inhibitory
concentrations (MICs) using the method described in the guidelines of NCCLS Document M27-A210 and
our previous paper.6 The results are listed in Table 1. The susceptibility assays were determined by the
broth dilution method performed in sterile flat-bottom 96-well microplates (Thermo Scientific, Waltham,
USA) as described previously in NCCLS guidelines, M-27 A document (NCCLS, 2002). Briefly, C.
albicans was inoculated at 35 °C and observed at 24 and 48 h. Five colonies greater than 1 mm in
diameter were selected, suspended in saline solution and adjusted to a final concentration of 0.5 x 103 to
2.5 x 103 in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) buffered to pH 7.0 with 0.165M
3-morpholinepropanesulfonic acid (MOPS; DOJINDO Laboratories, Kumamoto Japan). S. cerevisiae was
inoculated at 28 °C and observed at 24 and 48 h. Five colonies greater than 1 mm in diameter were
selected, suspended in saline solution and adjusted to a final concentration of 0.5 x 103 to 2.5 x 103 in YM
medium (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). The antifungal agents
itraconazole and miconazole (Wako Pure Chemical Industries, Osaka, Japan), were used as positive
control in the susceptibility tests. Itraconazole and miconazole were dissolved in DMSO. The drugs were
prepared at the 1280 µg/mL concentration in DMSO. The drug solutions were diluted in RPMI medium
or YM medium and final drugs concentrations ranged from 128 to 0.02 µg/mL After 48 h of incubation at
35 °C, MIC was determined visually by comparing its turbidity with the drug-free growth control well.
The MIC values were defined as the lower drug concentration, which resulted in reduction of 80% in the
turbidity in comparison with the drug-free growth control well.
ACKNOWLEDGEMENTS
The authors thank Hiroshi Hanazono and Yukiko Iwase for obtaining MS and NMR spectra and Junko
Honda for her valuable help with elemental analyses.
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