Synthesis and biological activity of some new pyrazole copper(II) complexes

Article abstract of DOI:10.1002/jhet.919

As a part of systematic investigation of synthesis and biologically active compounds of pyrazole derivatives containing transition metal, several new pyrazole copper(II) complexes 3a-f were synthesized from pyrazole sodium salts 2a-f, which were produced from spiro-pyrazoles 1a-f and sodium hydride by a ring-opening reaction. All the synthesized compounds were characterized by spectroscopic analysis. Pyrazole copper(II) complexes 3a-d and 3f exhibited high DNA cleavage activity in vitro. Furthermore, compounds 3a-f were tested for their growth inhibitory activity in A549 lung cancer, B16F10 murine melanoma, and HeLa human uterine carcinoma cells. Compounds 3c,d displayed moderate B16F10 and HeLa inhibitory activity levels (3c: IC₅₀ = 45 μM in B16F10 cells and 34 μM in HeLa cells, 3d: IC₅₀ = 50 μM in B16F10 cells and 32 μM in HeLa cells).

Full text of DOI:10.1002/jhet.919

1218
Synthesis and Biological Activity of Some New Pyrazole
Copper(II) Complexes
Vol 49
*
Hiroshi Maruoka, Sho Nishida, Nobuhiro Kashige, Yuki Yoshimura,
Masahiko Omori, Ryoko Tomita, Eiichi Masumoto, Fumi Okabe,
Fumio Miake, 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 December 15, 2010
DOI 10.1002/jhet.919
Published online 26 October 2012 in Wiley Online Library (wileyonlinelibrary.com).
As a part of systematic investigation of synthesis and biologically active compounds of pyrazole
derivatives containing transition metal, several new pyrazole copper(II) complexes 3aÀf were synthesized
from pyrazole sodium salts 2aÀf, which were produced from spiro-pyrazoles 1aÀf and sodium hydride
by a ring-opening reaction. All the synthesized compounds were characterized by spectroscopic analysis.
Pyrazole copper(II) complexes 3aÀd and 3f exhibited high DNA cleavage activity in vitro. Furthermore,
compounds 3aÀf were tested for their growth inhibitory activity in A549 lung cancer, B16F10 murine
melanoma, and HeLa human uterine carcinoma cells. Compounds 3c,d displayed moderate B16F10
and HeLa inhibitory activity levels (3c: IC50 = 45 μM in B16F10 cells and 34 μM in HeLa cells,
3d: IC50 = 50 μM in B16F10 cells and 32 μM in HeLa cells).
J. Heterocyclic Chem., 49, 1218 (2012).
INTRODUCTION
Bleomycin is a glycopeptide containing several unusual
amino acids, sugars, a pyrimidine, and a planar bithiazole
ring system. The bleomycin molecule may be formally
divided between its DNA-binding domain and its metal-
chelating domain. Bleomycin is believed to act by
degrading cellular DNA in a reaction requiring iron(II)
and oxygen cofactors [11–14]. The evidence strongly
suggests that the ultimate agent of DNA damage is some
form of activated reduced oxygen, produced as a conse-
quence of oxidation of bleomycin-chelated iron(II) to
iron(III) in a quaternary DNA-bleomycin-iron-oxygen
complex. On the other hand, copper(II) complexes are
known to be effective DNA interaction and cleavage
agents [15–22]. These complexes are structurally well-
defined and thus suitable for mechanistic studies. Metal
coordination complexes have been described as good
models of many natural enzymes that mediate this impor-
tant cleavage reaction.
The cleaving agents of nucleic acid have attracted
extensive attention due to their potential applications in
the fields of molecular biological technology and drug
development [1,2]. DNA is an important cellular receptor
and many chemicals exert their antitumor effects through
binding to DNA thereby changing the replication of
DNA and inhibiting the growth of tumor cells [3,4]. Then
discussing the mechanism of compounds cleaving and/or
binding to DNA possesses significant meanings. As well-
known, the interaction of small molecular ligands and its
transition metal complexes with DNA has been the main
subject of intense investigations for almost half of a
century [5]. Many transition metals, such as copper, zinc,
and nickel, are important trace metals in human body.
There has been considerable interest in developing new
synthetic reagents to manipulate DNA, with selectivity
different from the naturally occurring system. Simple metal
complexes have been successfully employed to accelerate
the rate of double-stranded DNA hydrolysis and those
metal complexes with intrinsically high affinity for DNA
are the most effective reagent [6–10]. In this context, the
design of small complexes that can bind to DNA becomes
more and more important.
Pyrazole derivatives are also well established in the
literatures as important biologically effective heterocyclic
compounds. These derivatives are the subject of many re-
search studies due to their widespread potential pharma-
cological activities such as analgesic, antidepressant,
antibacterial, plant growth regulatory, anti-inflammatory,
and antihyperglycemic activities [23–28]. Hence, the
© 2012 HeteroCorporation

September 2012
Synthesis and Biological Activity of Some New Pyrazole Copper(II) Complexes
1219
in N,N-dimethylformamide was stirred at room temperature
for 1 h and then the solvent wad removed in vacuo, the
expected pyrazole sodium salts 2aÀf were isolated by the re-
crystallization in good yields. The results are summarized in
Table 1. Elemental analyses and spectral data of 2aÀf are
consistent with the assigned structures (see “Experimental”
section).
Figure 1. Structures of pyrazole metal complexes A and B.
In the next step, a metal exchange reaction of pyrazole
sodium salts 2aÀf was examined. After some optimization,
we found the reaction condition under which pyrazole
copper(II) complexes 3aÀf could be isolated in the
presence of copper(II) acetate. When a mixture of 2aÀf
and copper(II) acetate monohydrate in methylene chloride
and water was stirred at room temperature for 24 h,
pyrazole copper(II) complexes 3aÀf were obtained in
moderate yields (Scheme 2 and Table 2). By comparison
of the IR, NMR, mass spectra, and elemental analyses of
3aÀf it seems that the structural assignments given to these
compounds are correct (see Experimental section). For ex-
ample, the IR spectrum of 3a displays bands at 3447 cmÀ1
due to a hydroxyl group and at 1717 cmÀ1 due to two ester
carbonyl groups. The 1H NMR spectrum of 3a in deuterio-
chloroform exhibits two three-proton singlets at δ 1.85
attributable to two methyl protons and two three-proton
singlets at δ 3.58 due to two methyl ester protons. The
13C NMR spectrum of 3a shows a signal at δ 13.6 due to
two methyl carbons, a signal at δ 51.8 due to two methyl
carbons of the methyl ester function, two signals at
δ 132.5 and 159.6 due to the olefin carbons, and a signal
at δ 170.4 due to two carbonyl carbons of the methyl ester
function.
development of improved methods for the synthesis of
substituted pyrazole derivatives has acquired relevance
to current research. Furthermore, the coordination chem-
istry of pyrazole derivatives such as 4-acyl-pyrazolones
A, wherein M represents Cu(II), Zn(II), Co(II), Ni(II),
Mn(II), Mg(II), or Hg(II), is well developed with respect
to their wide spectrum of applications, e.g., metal ex-
tractants, NMR shift reagents, and in laser technology
[29–36] (Fig. 1). In connection with our current research
interests in the synthesis and reactivity of pyrazole deriva-
tives [37,38], we have reported the synthesis of pyrazole
tin(IV) complexes B [39]. Based on these properties, it
can be reasonably supposed that the development of
synthetic strategies for new pyrazole metal complexes
might provide additional lead molecules for drug discov-
ery. For these reasons, we have been interested in the
preparation of pyrazole copper(II) complexes to evaluate
their biological activity and now report the results of our
investigation, in vitro their DNA cleavage and antitumor
activities.
RESULTS AND DISCUSSION
Finally, we have tested in vitro DNA cleavage activity
of all the synthesized compounds 2aÀf and 3aÀf. The
values obtained for activity were based on the remaining
amounts of covalently closed circular duplex DNA of
plasmid pBR322 (ccc-DNA) [40–42]. The DNA cleavage
activity data are summarized in Table 3. The activity is
accelerated to a remarkable degree by the addition of
cupric ion (Cu2+), which may stimulate the production of
active radicals such as hydroxyl radical, resulting in DNA
damage. Unfortunately, it was found that pyrazole sodium
salts 2aÀf exhibited no DNA damage, with or without
Cu2+ (entries 2À7). Interestingly, in the absence of Cu2+,
Initially, the synthesis of pyrazole sodium salts 2aÀf has
been accomplished as outlined in Scheme 1. In earlier
studies, we have demonstrated the ring-opening reaction
[38] of spiro-pyrazole compounds, which were prepared
by treatment of 2,4-dihydro-5-methyl-2-phenyl-4-(di-
phenylmethylene)-3H-pyrazol-3-one and α-chloro esters
according to our previous investigation [37]. Based on the
ring-opening reaction, we hypothesized if the spiro-pyrazole
compounds would be treated with an appropriate basic con-
dition, the isolation of pyrazole sodium salts would be possi-
ble through a ring-opening reaction. Hence, we tried the
synthesis of pyrazole sodium salts 2aÀf. In fact, when a mix-
ture of spiro-pyrazole compounds 1aÀf and sodium hydride
Table 1
Synthesis of pyrazole sodium salts 2a‐f.
Scheme 1
Entry
Substrate
Product
Yield (%)
1
2
3
4
5
6
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
99
99
93
99
99
99
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H. Maruoka, S. Nishida, N. Kashige, Y. Yoshimura, M. Omori, R. Tomita, E. Masumoto, F. Okabe, Vol 49
F. Miake, K. Yamagata, and T. Fujioka
Scheme 2
pyrazole copper(II) complexes 3aÀd and 3f showed mod-
erate activity (entries 8À11, 13, and Fig. 2) and these activ-
ities were obviously accelerated by addition of 1 mM Cu2+
(entries 8À11, 13, and Fig. 3).
chemistry. Further studies on the synthesis of new substi-
tuted pyrazole metal complexes are under way.
EXPERIMENTAL
On the basis of the above results, to elucidate in vitro the
DNA damage activity of pyrazole copper(II) complexes
3aÀf, the antitumor activity of 3aÀf was measured in
A549 lung cancer, B16F10 murine melanoma, and HeLa
human uterine carcinoma cells using MTT assay [43,44].
In general, 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. The growth inhibitory properties
(IC50) for the compounds 3aÀf are listed in Table 4.
Interestingly, the data obtained by MTT assay showed that
all the synthesized compounds 3aÀf had inhibitory effects
on the growth of B16F10 and HeLa cells in dosage-dependent
manners. However, compounds 3aÀf could not almost inhibit
the cell growth of A549 at 100 μM after 48 h of the treatment.
Taken altogether, pyrazole copper(II) complex 3d was the
most potent compound in this series, having an IC50 value of
32 μM in suppressing HeLa cell growth (entry 4).
In conclusion, we have demonstrated a novel method for
the synthesis of pyrazole copper(II) complexes 3aÀf,
proceeding by a metal exchange reaction when pyrazole
sodium salts 2aÀf were treated with copper(II) acetate.
Furthermore, we found that compounds 3aÀf could
suppress B16F10 and HeLa cancer cell growth. Pyrazole
copper(II) complex 3d was the most effective small mole-
cule in inhibiting HeLa cell growth and might perform its
action through including DNA damage. Functionalized
pyrazole metal complexes are important for the preparation
of biologically active compounds with interest in medicinal
All melting points are uncorrected. The IR spectra were
recorded on a JASCO FT/IR-4100 spectrometer. The 1H and
13C NMR spectra were recorded on a JEOL JNM-A500 spec-
trometer at 500 and 125 MHz, respectively. The 1H and 13C
chemical shifts (δ) are reported in parts per million (ppm) relative
Table 3
DNA cleavage by 2a‐f and 3a‐f in the absence and/or presence of Cu2+
.
Relative amounts of DNA (%)
DNA
Entry Compound
type
Without Cu2+a
With Cu2+b
1
Controlc
2ad
2bd
2cd
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
ccc‐
Oc‐
100
0
100
0
97
3
95
5
99
1
100
0
97
3
74
26
52
48
56
44
52
48
94
6
100
0
100
0
99
1
98
2
100
0
100
0
100
0
60
40
31
69
28
72
28
72
100
0
2
3
4
5
2dd
2ed
6
7
2fd
8
3ae
9
3be
3ce
10
11
12
13
3de
3ee
Table 2
3fe
75
25
71
29
Synthesis of pyrazole copper(II) complexes 3a‐f.
Entry
Substrate
Product
Yield (%)
aIncubation for 3 h.
bIncubation for 1 h.
cAmount: 0 mM.
dAmount: 1 mM.
eAmount: 0.1 mM.
1
2
3
4
5
6
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
88
72
68
48
60
74
As activity was accelerated upon addition of Cu2+, the quantity of com-
pounds and the incubation time were minimized until differences in ac-
tivity could be observed.
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September 2012
Synthesis and Biological Activity of Some New Pyrazole Copper(II) Complexes
1221
Table 4
Antitumor activity of 3a‐f against A549, B16F10, and HeLa cells in vitro.
IC50 values (μM)
Entry
Compound
A549
>100
No effect
>100
86
B16F10
HeLa
1
2
3
4
5
6
3a
3b
3c
3d
3e
3f
65
70
45
50
69
92
36
36
34
32
36
51
90
>100
Figure 2. DNA cleavage by 3a–f in the absence of Cu2+
.
to tetramethylsilane as internal standard. The positive FAB mass
spectra were obtained on a JEOL JMS-700T spectrometer. The
elemental analyses were performed on a YANACO MT-6 CHN
analyzer.
Ph─H); 13C NMR (deuterium oxide): δ 15.6, 15.7 (pyrazole
3ÀMe, CO2CH2Me), 64.9 (CO2CH2Me), 102.5 (pyrazole
CÀ4), 125.1, 128.2 (Ph─C), 128.6 (Ph2C═C─CO2CH2Me),
130.5, 130.8, 131.0, 131.2, 131.8, 132.2, 133.4, 142.2,
145.1, 145.7 (Ph─C), 149.7 (Ph2C═C─CO2CH2Me), 152.1
(pyrazole CÀ3), 164.5 (pyrazole CÀ5), 177.0 ppm (CO); ms: m/z
447 [M+H]+. Anal. Calcd. for C27H23N2O3Na·0.1H2O: C, 72.05;
H, 5.24; N, 6.22. Found: C, 72.05; H, 5.53; N, 6.18.
Isopropyl 2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
3,3-diphenylacrylate sodium salt (2c). This compound was
obtained as colorless needles, mp 216À218°C; IR (potassium
bromide): n 1695 cmÀ1 (CO); 1H NMR (deuterium oxide): δ
0.97 (d, J = 6.4 Hz, 6H, CO2CHMe2), 1.61 (s, 3H, pyrazole
3ÀMe), 4.85 (sep, J = 6.4 Hz, 1H, CO2CHMe2), 7.24À7.30 (m,
8H, Ph─H), 7.41À7.47 (m, 5H, Ph─H), 7.55À7.57 ppm (m, 2H,
Ph─H); 13C NMR (deuterium oxide): δ 15.6 (pyrazole 3ÀMe), 23.3
(CO2CHMe2), 73.1 (CO2CHMe2), 102.4 (pyrazole CÀ4), 125.1,
128.2 (Ph─C), 129.0 (Ph2C═C─CO2CHMe2), 130.5, 130.8, 131.0,
131.2, 131.8, 132.4, 133.4, 142.2, 145.2, 145.7 (Ph─C), 149.0
(Ph2C═C─CO2CHMe2), 152.3 (pyrazole CÀ3), 164.5 (pyrazole
CÀ5), 176.6 ppm (CO); ms: m/z 461 [M+H]+. Anal. Calcd. for
C28H25N2O3Na·0.7H2O: C, 71.08; H, 5.62; N, 5.92. Found: C,
71.05; H, 5.70; N, 5.88.
General procedure for the preparation of pyrazole sodium salts
2aÀf from 1aÀf and sodium hydride. To an ice-cooled and stirred
solution of 1aÀf [37] (1 mmol) in N,N-dimethylformamide (5 mL)
was added 60% sodium hydride (0.04 g, 1 mmol). After the mixture
was stirred at room temperature for 1 h, the solvent was removed in
vacuo. The residue was recrystallized from chloroform-petroleum
ether to afford 2aÀf.
Methyl 2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3,
3-diphenylacrylate sodium salt (2a). This compound was
obtained as colorless needles, mp 200–202°C; IR (potassium
bromide): n 1699 cmÀ1 (CO); 1H NMR (deuterium oxide): δ 1.56
(s, 3H, pyrazole 3ÀMe), 3.56 (s, 3H, CO2Me), 7.23–7.30 (m, 9H,
Ph─H), 7.40À7.48 (m, 4H, Ph─H), 7.57–7.59 ppm (m, 2H,
Ph─H); 13C NMR (deuterium oxide): δ 15.5 (pyrazole 3ÀMe),
55.2 (CO2Me), 102.5 (pyrazole CÀ4), 125.1 (Ph─C), 128.1
(Ph2C═C─CO2Me), 128.2, 130.3, 130.6, 130.8, 131.3, 131.2,
131.7, 131.8, 132.2, 132.4, 133.3, 133.4, 142.1, 145.1, 145.6
(Ph─C), 150.2 (Ph2C═C─CO2Me), 152.0 (pyrazole CÀ3), 164.5
(pyrazole CÀ5), 177.4 ppm (CO); ms: m/z 433 [M+H]+. Anal.
Calcd. for C26H21N2O3Na·1H2O: C, 69.32; H, 5.15; N, 6.22.
Found: C, 69.36; H, 5.36; N, 6.10.
tert-Butyl
2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
3,3-diphenylacrylate sodium salt (2d). This compound was
obtained as colorless needles, mp 197À199°C; IR (potassium
Ethyl 2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3,3-
diphenylacrylate sodium salt (2b). This compound was obtained
as colorless needles, mp 76À79°C; IR (potassium bromide):
n 1705 cmÀ1 (CO); 1H NMR (deuterium oxide): δ 0.92
(t, J = 7.3 Hz, 3H, CO2CH2Me), 1.57 (s, 3H, pyrazole 3ÀMe),
4.01 (q, J = 7.3 Hz, 2H, CO2CH2Me), 7.22À7.29 (m, 8H,
Ph─H), 7.39À7.48 (m, 5H, Ph─H), 7.57À7.59 ppm (m, 2H,
1
bromide): n 1691 cmÀ1 (CO); H NMR (deuterium oxide): δ 1.21
(s, 9H, CO2CMe3), 1.70 (s, 3H, pyrazole 3ÀMe), 7.20À7.30 (m,
8H, Ph─H), 7.41À7.47 (m, 5H, Ph─H), 7.54À7.56 ppm (m, 2H,
Ph─H); 13C NMR (deuterium oxide): δ 15.6 (pyrazole 3ÀMe), 29.7
(CO2CMe3), 86.0 (CO2CMe3), 102.4 (pyrazole CÀ4), 125.2, 128.2
(Ph─C), 130.0 (Ph2C═C─CO2CMe3), 130.3, 130.7, 130.8, 131.2,
131.7, 132.5, 133.1, 142.2, 145.3, 145.8 (Ph─C), 149.3
(Ph2C═C─CO2CMe3), 152.4 (pyrazole CÀ3), 164.3 (pyrazole
CÀ5), 175.8 ppm (CO); ms: m/z 475 [M+H]+; high-resolution
ms: Calcd. for C29H28N2O3Na 475.1998, found 475.1991.
2-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3,3-
diphenylacrylonitrile sodium salt (2e). This compound was
obtained as colorless needles, mp >280°C; IR (potassium
bromide): n 2216 cmÀ1 (CN); 1H NMR (deuterium oxide): δ
1.65 (s, 3H, pyrazole 3ÀMe), 7.20À7.32 (m, 6H, Ph─H),
7.46À7.50 (m, 7H, Ph─H), 7.57À7.59 ppm (m, 2H, Ph─H); 13C
NMR (deuterium oxide): δ 15.3 (pyrazole 3ÀMe), 98.5 (pyrazole
CÀ4), 106.2 (CN), 123.7 (Ph2C═C─CN), 125.4, 128.5, 131.0,
131.3, 131.6, 131.8, 132.3, 132.8, 132.9, 141.9, 142.9, 143.4
(Ph─C), 151.3 (pyrazole CÀ3), 160.1 (Ph2C═C─CN), 164.0
ppm (pyrazole CÀ5); ms: m/z 400 [M+H]+. Anal. Calcd. for
Figure 3. DNA cleavage by 3a–f in the presence of Cu2+
.
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H. Maruoka, S. Nishida, N. Kashige, Y. Yoshimura, M. Omori, R. Tomita, E. Masumoto, F. Okabe, Vol 49
F. Miake, K. Yamagata, and T. Fujioka
C25H18N3ONa·2.7H2O: C, 67.01; H, 5.26; N, 9.38. Found: C,
66.91; H, 4.97; N, 9.27.
bromide):
n
3439 (OH), 1708 cmÀ1 (CO); 1H NMR
(deuteriochloroform): δ 0.98À1.05 (m, 12H, 2× CO2CHMe2), 1.85
(s, 6H, 2× pyrazole 3ÀMe), 4.94 (br s, 2H, 2× CO2CHMe2),
7.16À7.53 (m, 27H, Ph─H and OH), 8.00 ppm (br s, 4H, Ph─H);
13C NMR (deuteriochloroform): δ 13.6, 13.7 (2× pyrazole 3ÀMe),
21.1, 21.3, 21.48, 21.54 (2× CO2CHMe2), 68.5 (2× CO2CHMe2),
118.5, 118.8, 122.0, 122.3, 124.2, 125.0, 127.3, 127.8, 128.0,
128.1, 128.2, 128.3, 128.4 (Ph─C), 128.5 (2× pyrazole CÀ4),
128.9, 129.0, 129.1, 129.5, 129.8, 130.5 (Ph─C), 132.6 (2×
Ph2C═C─CO2CHMe2), 138.6, 140.5 (Ph─C), 144.9 (2× pyrazole
CÀ3), 159.4 (pyrazole CÀ5), 159.7 (2× Ph2C─C─CO2CHMe2),
161.7 (pyrazole CÀ5), 170.4 ppm (2× CO); ms: m/z 939 [M+H]+.
Anal. Calcd. for C56H51N4O6Cu·1.2H2O: C, 69.98; H, 5.60; N, 5.83.
Found: C, 70.04; H, 5.32; N, 5.75.
N,N-Diethyl-2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3,
3-diphenylacrylamide sodium salt (2f). This compound was obtained
as colorless needles, mp 169À172°C; IR (potassium bromide): n 1674
cmÀ1 (CO); 1H NMR (deuterium oxide): δ 0.77, 0.99 [t, J = 7.3 Hz,
6H, CON(CH2Me)2], 1.88 (s, 3H, pyrazole 3ÀMe), 3.07, 3.36, 3.54,
3.73 [br s, 4H, CON(CH2Me)2], 7.22À7.27 (m, 8H, Ph─H),
7.36À7.37 (m, 3H, Ph─H), 7.40À7.47 ppm (m, 4H, Ph─H); 13C
NMR (deuterium oxide): δ 13.8, 14.2 [CON(CH2Me)2], 15.8
(pyrazole 3ÀMe), 41.5, 46.1 [CON(CH2Me)2], 101.8 (pyrazole
CÀ4), 118.4, 125.5, 128.3, 129.9, 130.6, 131.0 (Ph─C), 131.4
[Ph2C═C─CON(CH2Me)2], 131.7, 132.4, 133.4, 142.2, 145.4
(Ph─C), 145.6 (pyrazole CÀ3), 152.5 [Ph2C═C─CON(CH2Me)2],
163.3 (pyrazole CÀ5), 176.2 ppm (CO); ms: m/z 474 [M+H]+.
Anal. Calcd. for C29H28N3O2Na·0.7H2O: C, 71.65; H, 6.10; N,
8.64. Found: C, 71.68; H, 6.23; N, 8.69.
General procedure for the preparation of pyrazole copper(II)
complexes 3aÀf from 2aÀf and copper(II) acetate. To a stirred
solution of 2aÀf (1 mmol) in methylene chloride (10 mL) and
water (10 mL) was added copper(II) acetate monohydrate
(0.10 g, 0.5 mmol). After the mixture was stirred at room
temperature for 24 h, the precipitate was removed by filtration
and washed with methylene chloride. The combined filtrates
were extracted with methylene chloride (60 mL). The extract
was dried over anhydrous sodium sulfate and concentrated
in vacuo. The crude products 3aÀf were sufficiently pure to be
used without further purification.
tert-Butyl 2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
3,3-diphenylacrylate copper(II) complex (3d). This compound
was obtained as brown solid, mp 85À88°C; IR (potassium
bromide): n
3446 (OH), 1716 cmÀ1 (CO); 1H NMR
(deuteriochloroform): δ 1.26 (s, 18H, 2× CO2CMe3), 1.86 (s, 6H,
2× pyrazole 3ÀMe), 2.94 (s, 1H, OH), 7.16À7.53 (m, 26H,
Ph─H), 8.00À8.02 ppm (m, 4H, Ph─H); 13C NMR
(deuteriochloroform): δ 13.6 (2× pyrazole 3ÀMe), 27.5, 27.6, 27.7,
27.9 (2× CO2CMe3), 71.5 (2× pyrazole CÀ4), 81.9 (2× CO2CMe3),
118.5, 118.7, 122.2, 124.0, 124.9, 127.8, 127.9, 128.0, 128.1,
128.2, 128.3, 128.4, 128.7, 128.8, 128.9, 129.1, 129.5, 129.6, 129.8
(Ph─C), 132.8 (2× Ph2C═C─CO2CMe3), 138.6, 140.4 (Ph─C),
144.9 (2× pyrazole CÀ3), 158.7 (pyrazole CÀ5), 159.8 (2×
Ph2C═C─CO2CMe3), 161.4 (pyrazole CÀ5), 170.4 ppm
(2× CO); ms: m/z 967 [M+H]+. Anal. Calcd. for
C58H55N4O6Cu·1H2O: C, 70.68; H, 5.83; N, 5.68. Found:
C, 70.64; H, 5.99; N, 5.59.
Methyl 2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3,
3-diphenylacrylate copper(II) complex (3a). This compound was
obtained as brown solid, mp 107À110°C; IR (potassium
bromide):
n
3447 (OH), 1717 cmÀ1 (CO); 1H NMR
2-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
3,3-diphenylacrylonitrile copper(II) complex (3e). This compound
was obtained as brown solid, mp 157À160°C; IR (potassium
(deuteriochloroform): δ 1.85 (s, 6H, 2× pyrazole 3ÀMe), 3.28 (br
s, 1H, OH), 3.58 (s, 6H, 2× CO2Me), 7.10À7.54 (m, 26H, Ph─H),
7.96À7.98 ppm (m, 4H, Ph─H); 13C NMR (deuteriochloroform):
δ 13.6 (2× pyrazole 3ÀMe), 51.8 (2× CO2Me), 118.5 (2× pyrazole
CÀ4), 119.2, 122.3, 124.4, 125.2, 127.7, 127.8, 127.9, 128.0, 128.1,
128.2, 128.3, 128.4, 128.9, 129.1, 129.2, 129.3, 129.4, 129.9
(Ph─C), 132.5 (2× Ph2C═C─CO2Me), 138.4, 140.6 (Ph─C),
144.7 (2× pyrazole CÀ3), 159.4 (pyrazole CÀ5), 159.6 (2×
Ph2C═C─CO2Me), 162.9 (pyrazole CÀ5), 170.4 ppm (2× CO);
ms: m/z 883 [M+H]+. Anal. Calcd. for C52H43N4O6Cu·1H2O: C,
69.28; H, 5.03; N, 6.21. Found: C, 69.27; H, 4.78; N, 6.15.
bromide):
n
3438 (OH), 2209 cmÀ1 (CN); 1H NMR
(deuteriochloroform): δ 1.93, 1.94, 1.95, 2.26 (s, 6H, 2× pyrazole
3ÀMe), 3.18 (br s, 1H, OH), 7.00À7.97 ppm (m, 30H, Ph─H);
13C NMR (deuteriochloroform): δ 13.6, 13.7 (2× 3ÀMe), 78.9
(2×pyrazole CÀ4), 114.4, 115.1 (2× CN), 118.5, 119.1, 122.8,
123.0, 124.3, 125.3, 125.7, 128.0, 128.2, 128.3, 128.4, 128.5,
128.6, 128.8, 128.9, 129.0, 129.1, 129.2, 129.3, 129.4, 129.5,
129.7, 130.0, 130.1, 130.2, 130.2, 130.5, 130.6, 130.9 (Ph─C),
135.9 (2× Ph2C═C─CN), 136.5, 137.1, 137.9, 138.8, 139.0
(Ph─C), 140.8, 141.7 (2× pyrazole CÀ3), 156.9, 158.4 (2×
Ph2C═C─CN), 162.6, 163.1 ppm (2× pyrazole CÀ5); ms: m/z 817
[M+H]+. Anal. Calcd. for C50H37N6O2Cu·0.7H2O: C, 72.35; H,
4.66; N, 10.12. Found: C, 72.40; H, 4.70; N, 10.01.
Ethyl 2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3,3-
diphenylacrylate copper(II) complex (3b). This compound was
obtained as brown solid, mp 90À93°C; IR (potassium bromide):
1
n 3446 (OH), 1716 cmÀ1 (CO); H NMR (deuteriochloroform):
δ 0.98 (t, J = 7.0 Hz, 6H, 2× CO2CH2Me), 1.85 (s, 6H, 2× pyrazole
3ÀMe), 3.82 (br s, 1H, OH), 4.02À4.08 (m, 4H, 2× CO2CH2Me),
7.06À7.55 (m, 26H, Ph─H), 7.97À8.00 ppm (m, 4H, Ph─H); 13C
NMR (deuteriochloroform): δ 13.6, 13.7 (2× pyrazole 3ÀMe, 2×
CO2CH2Me), 60.8 (2× CO2CH2Me), 118.9, 122.3, 124.3, 125.1,
127.6, 127.8, 127.9, 128.1 (Ph─C), 128.2 (2× pyrazole CÀ4),
128.9, 129.0, 129.1, 129.2, 129.9 (Ph─C), 132.6 (2×
Ph2C═C─CO2CH2Me), 138.5, 140.6 (Ph─C), 144.8 (2× pyrazole
CÀ3), 159.5 (pyrazole CÀ5, 2× Ph2C═C─CO2CH2Me), 162.3
(pyrazole CÀ5), 170.4 ppm (2× CO); ms: m/z 911 [M+H]+. Anal.
Calcd. for C54H47N4O6Cu: C, 71.15; H, 5.20; N, 6.15. Found: C,
71.43; H, 5.17; N, 6.10.
N,N-Diethyl-2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
3,3-diphenylacrylamide copper(II) complex (3f). This compound
was obtained as brown solid, mp 171À174°C; IR (potassium
bromide):
n
3446 (OH), 1657 cmÀ1 (CO); 1H NMR
(deuteriochloroform): δ 0.55À1.27 [m, 12H, 2× CON(CH2Me)2],
1.62, 1.68 (s, 6H, 2× pyrazole 3ÀMe), 2.20À3.50 [m, 9H, OH
and 2× CON(CH2Me)2], 6.86À7.97 ppm (m, 30H, Ph─H); 13C
NMR (deuteriochloroform): δ 11.8, 13.2 [2× CON(CH2Me)2],
14.1 (2× 3ÀMe), 38.0, 42.3 [2× CON(CH2Me)2], 73.0 (2× pyrazole
CÀ4), 118.9, 122.2, 122.5, 125.0, 127.3, 127.4, 127.5, 127.7, 127.8,
127.9, 128.1, 128.2, 128.4, 128.5, 128.6, 128.7, 128.8, 128.9, 129.2,
129.4, 129.5, 129.6, 129.8, 130.1, 130.2 (Ph─C) 132.7, 133.5
[2× Ph2C═C─CON(CH2Me)2], 138.3, 140.0, 143.8 (Ph─C),
145.8 (2× pyrazole CÀ3), 161.0 [pyrazole CÀ5, 2×
Ph2C═C─CON(CH2Me)2], 165.0 (pyrazole CÀ5), 169.9 ppm
Isopropyl 2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
3,3-diphenylacrylate copper(II) complex (3c). This compound
was obtained as brown solid, mp 115À118°C; IR (potassium
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2012
Synthesis and Biological Activity of Some New Pyrazole Copper(II) Complexes
1223
(2× CO); ms: m/z 965 [M+H]+. Anal. Calcd. for C58H57N6O4Cu: C,
72.14; H, 5.95; N, 8.70. Found: C, 72.34; H, 6.12; N, 8.70.
DNA cleavage activity assay. The method of assaying the
DNA cleavage activity, using a covalently closed circular
duplex DNA of plasmid pBR322 (ccc-DNA) as a substrate, was
described in our previous investigation [40–42].
Tumor cell culture and MTT assay. A549 and HeLa cells
were obtained from DS Pharma Biomedical (Japan). B16F10
cells were purchased from American Type Culture Collection.
Tumor cells were cultured in Roswell Park Memorial Institute
1640 medium or Dulbecco’s Modified Eagle Medium at 37°C
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 [43] and our previous methods [44].
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet