Synthesis, structural characterization and biological activity of Cu(II) compounds incorporating pyrazole-derived ligand: Effect on cell growth in human colorectal carcinoma

Article abstract of DOI:10.1002/jccs.201100396

A series of Cu(II) compounds containing neutral multi-dentate ligand [2,6-diisopropylphenyl]-bis[(1-Hpyrazol-1-yl)methyl]amine (L₁) and pyrazole dimethoxethyl ligand [(1-H-pyrazol-1-yl)methyl]-bis(2-methoxyethyl) amine (L₂) were synthesized. Reactions of L₁ and L ₂ with copper(II) chloride generate L₁CuCl₂ (1) and L₂CuCl₂ (2), respectively. Compounds 1 and 2 have been characterized by elemental analysis and X-ray single crystal diffractometry. The effects of compounds 1 and 2 on the cell viability of various human cancer cells (including A549, COLO 205, HT-29, Hep3B, HepG2, Huh7, and PCL5 cells) were investigated. The results indicate that compound 2 has a strong inhibitory effect on cell growth in human colorectal carcinoma cells (COLO 205 cells and HT-29 cells).

Full text of DOI:10.1002/jccs.201100396

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis, Structural Characterization and Biological Activity of Cu(II) Compounds
Incorporating Pyrazole-derived Ligand: Effect on Cell Growth in Human Colorectal
Carcinoma
Chen-Yi Li,a Yu-Ling Lien,a Amitabha Datta,a Wan-Ting Chiu,a Chin-Lin Hsu,b,*
Jui-Hsien Huang,a,* Lance Horng,c Chia-Her Lind and Chi-Jung Sue
aDepartment of Chemistry, National Changhua University of Education, Changhua 50058, Taiwan
bSchool of Nutrition, Chung Shan Medical University, Taichung 402, Taiwan and Department of Nutrition,
Chung Shan Medical University Hospital, Taichung 402, Taiwan
cDepartment of Physics, National Changhua University of Education, Changhua 50058, Taiwan
dDepartment of Chemistry, Chung-Yuan Christian University, Chun-Li 320, Taiwan
eDepartment of Applied Chemistry, Chung Shan Medical University, Taichung 40201, Taiwan
(Received: Jun. 24, 2011; Accepted: Dec. 12, 2011; Published Online: Mar. 13, 2012; DOI: 10.1002/jccs.201100396)
A series of Cu(II) compounds containing neutral multi-dentate ligand [2,6-diisopropylphenyl]-bis[(1-H-
pyrazol-1-yl)methyl]amine (L1) and pyrazole dimethoxethyl ligand [(1-H-pyrazol-1-yl)methyl]-bis(2-
methoxyethyl)amine (L2) were synthesized. Reactions of L1 and L2 with copper(II) chloride generate
L1CuCl2 (1) and L2CuCl2 (2), respectively. Compounds 1 and 2 have been characterized by elemental
analysis and X-ray single crystal diffractometry. The effects of compounds 1 and 2 on the cell viability of
various human cancer cells (including A549, COLO 205, HT-29, Hep3B, HepG2, Huh7, and PCL5 cells)
were investigated. The results indicate that compound 2 has a strong inhibitory effect on cell growth in hu-
man colorectal carcinoma cells (COLO 205 cells and HT-29 cells).
Keywords: Copper; Pyrazole; Carcinoma.
INTRODUCTION
The increasing interest in chelating polydentate nitro-
of the cell cycle and induction of apoptosis could provide a
therapeutic strategy for the treatment of cancer.8 Pro-
grammed cell death plays an important role in the regula-
tion of cellular homeostasis.9 In cells responsive to apo-
ptotic stimuli, there are two major apoptotic pathways that
involve intrinsic (mitochondrion-dependent pathway) and
extrinsic (death receptor-dependent pathway) signaling
pathways.10 Marín-Hernández et al.11 indicated that some
mixed chelate copper-based drugs have potent antitumor
activity than cisplatin in in vivo and in vitro studies of a
variety of tumor cells. However, human cancer cell lines
are a useful model to study cell growth inhibition of tumor
cells by natural compounds or newly-synthesized com-
pounds.
gen-donor ligands, especially heterocyclic compounds, is
based on the occurrence of such systems in nature.1 Most
attention is given to model systems containing imidazoles,
benzimidazoles, and pyridines.2 In this connection, atten-
tion is also paid to pyrazole derivative,3 because of the sim-
ilarity of pyrazoles and imidazoles due to their significant
involvement in numerous stoichiometric and catalytic reac-
tions in the development of organometallic chemistry. Pyr-
azoles have been extensively used as N-donor ligands to
metals.4 The structure and properties of copper pyrazolates
are of significant interest.5 These compounds have been
found to exhibit a variety of structures with exo-bidentate
coordination of the pyrazolate ligand to the copper cen-
ters.6 Thus, the coordination chemistry of copper incorpo-
rating pyrazole-based ligands have drawn attention be-
cause of their interesting unusual structural features, re-
markable physical and chemical properties and biological
activity.7
We now develop our research using new pyrazolyl
ligands, [2,6-diisopropylphenyl]-bis[(1-H-pyrazol-1-yl)-
methyl]amine (L1) and pyrazole dimethoxethyl ligand
[(1-H-pyrazol-1-yl)methyl]-bis(2-methoxyethyl)amine
(L2) and their corresponding Cu(II) derivatives, L1CuCl2
(1) and L2CuCl2 (2). The aim of the present study has been
to investigate the effect of compounds 1 and 2 on the cell
The control of tumor cell proliferation by inhibition
* Corresponding author. E-mail: juihuang@cc.ncue.edu.tw (J.-H. Huang) and clhsu@csmu.edu.tw (C.-L. Hsu)
696
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Cu(II) Compounds Effect on Colorectal Carcinoma Cell Growth
Scheme II Cu-complexation with L1 and L2
viability of various human cancer cells.
RESULTS AND DISCUSSION
Synthesis and Characterization of the Compounds
[2,6-diisopropylphenyl]-bis[(1-H-pyrazol-1-yl)methyl]-
amine (L1) and [(1-H-pyrazol-1-yl)methyl]-bis(2-meth-
oxyethyl)amine (L2)
The ligands, [2,6-diisopropylphenyl]-bis[(1-H-pyr-
azol-1-yl)methyl]amine (L1) and [(1-H-pyrazol-1-yl)-
methyl]-bis(2-methoxyethyl)amine (L2), were prepared by
condensation between 2,6-diisopropylaniline or bis(2-
methoxyethyl)amine with pyrazole in presence of formal-
dehyde in ethanol (Scheme I). Both of the ligands L1 and L2
were isolated with satisfactory yield and used for com-
plexation without further purification.
Crystal Structures of L1, 1, and 2
Crystals of L1 suitable for X-ray diffraction analysis
were obtained from a toluene solution at -20 °C. The Sum-
mary of data collection and selected bond lengths and an-
gles are shown in Table 1 and 2, respectively. The molecu-
lar geometry of L1 is shown in Fig. 1. Apparently, the N(1)
nitrogen atom uses sp2 hybrid orbitals to overlap with the
orbitals of the phenyl carbon and two pyrazol-1-ly methyl
carbons and the lone pair electrons locating on the unhy-
brid p orbital of the N(1) nitrogen atom may delocalize to
the phenyl ring. This results the total bond angle around the
aniline nitrogen N(1) closes to 360° (118.13, 118.00, and
116.73).
Scheme I Formation of ligands L1 and L2
Crystals of 1 suitable for X-ray diffraction analysis
were obtained from a CH3CN solution at -20 °C. The mo-
lecular geometry of 1 is shown in Fig. 2. The geometry of 1
can be described as a highly distorted tetrahedral. The bond
angle of Cl(1)-Cu(1)-Cl(2) (131.23(5)°) is larger than
109.5°, presumably due to the large lone pair interaction of
the two chlorine atoms. The L1 ligand binds to copper atom
through N(1) and N(5) forming a eight-member ring with
the bond angle of N(1)-Cu(1)-N(5) at 129.99(13)°. The
The 1H and 13C NMR spectra of L1 show the methy-
1
lene fragments at d 5.49 and 69.6, respectively. The H
NMR spectrum of L2 is also consistent with the structure in
solution having one singlet appeared at d 3.28 for the two
methoxy groups. Two triplets appeared at d 2.76 and 3.45,
are assigned as the two methylene groups of the pendant
N(CH2CH2OMe)2 fragment and a singlet at d 5.05 is as-
signed as the methylene protons between pyrazole and
amine.
Syntheis of L1CuCl2 (1) and L2CuCl2 (2)
Reactions of L1 and L2 with one equivalent of anhy-
drous CuCl2 in acetonitrile or methylene chloride generate
L1CuCl2 (1) and L2CuCl2 (2), respectively in satisfactory
yield. Both of compounds 1 and 2 are paramagnetic com-
pounds and therefore no NMR spectra could be obtained.
Both of the compounds were characterized on the basis of
elemental analysis and X-ray structure determination.
Fig. 1. The molecular structure of L1. The atoms are
drawn with 30% probability ellipsoids and the
hydrogen atoms are omitted for clarity.
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Table 1. Crystal data and structure refinements for L1, 1 and 2 (2a and 2b)
L1
1
2a
2b
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
C20H27N5
337.47
150(2)
0.71073
Orthorhombic
Pbca
C20H27N5CuCl2
471.91
150(2)
0.71073
Trigonal
C42H76N13O8Cu4Cl8
1428.92
150(2)
0.71073
Monoclinic
C2/c
C10H19N3O2CuCl2
347.72
150(2)
0.71073
Monoclinic
P21/n
Space group
R-3
Unut cell dimensions
a = 13.6830(5) Å
b = 14.9767(6) Å
c = 18.8372(6) Å
a = 90°
a = 30.548(2) Å
b = 30.548(2) Å
c = 13.8507(10) Å
a = 90°
a = 31.379(2) Å
b = 12.6377(7) Å
c = 31.1777(18) Å
a = 90°
a = 6.91060(10) Å
b = 18.2887(3) Å
c = 11.4727(2) Å
a = 90°
b = 90°
g = 90°
b = 90°
g = 120°
b = 102.449(3)°
g = 90°
b = 96.7610 (10)°
g = 90°
Volume (Å3), Z
3860.2(2), 8
1.161
0.071
1456
11193.8(15), 18
1.260
1.106
4410
12072.9(13), 8
1.572
1.802
5880
1439.90(4), 4
1.604
1.885
716
Density (calculated) (Mg/m3)
Absorption coefficient (mm-1)
F(000)
Crystal size (mm3)
0.50 ´ 0.46 ´ 0.26
2.16 to 29.07°
42668
5156; Rint = 0.0340
0.9817 and 0.9651
5156 / 0 / 235
1.026
R1 = 0.0414
wR2 = 0.0995
R1 = 0.0627
wR2 = 0.1118
0.290 and -0.199
0.30 ´ 0.15 ´ 0.11
1.33 to 29.00°
80890
6596; Rint = 0.0679
0.8880 and 0.7325
6596 / 0 / 257
1.085
R1 = 0.0548
wR2 = 0.1908
R1 = 0.0904
wR2 = 0.2181
3.135 and -0.458
0.40 ´ 0.21 ´ 0.15
1.33 to 29.08°
129877
16164; Rint = 0.0584
0.7738 and 0.5327
16164 / 0 / 679
1.045
R1 = 0.0351
wR2 = 0.00738
R1 = 0.0672
wR2 = 0.0898
0.615 and -0.573
0.18 ´ 0.16 ´ 0.15
2.11 to 29.03°
16453
3840; Rint = 0.0259
0.7652 and 0.7278
3840 / 0 / 165
1.037
R1 = 0.0237
wR2 = 0.0635
R1 = 0.0288
wR2 = 0.0661
0.439 and -0.497
q range for data collection
Reflections collected
Independent reflections
Max. and min. transmission
Data/restraints/parameters
Goodness-of-fit on F2
Final Rindices
[I > 2s(I)]
R indices (all data)
Largest diff. peak and hole (e.Å-3)
aR1 = S½F0½-½Fc½/S½F0½; bwR2 = [S[w(F02 - Fc2)2] / S[w(F02 )2]1/2
.
bond length of Cu(1)-N(1) and Cu(1)-N(5) are 1.981(3)
and 1.997(3) Å, respectively.
a saturated CH3CN solution at -20 °C after reacting L2 with
CuCl2. Crystals of 2b were obtained in a similar procedure
excepting the volatiles were removed first after reaction
complete and then re-crystallized again from a saturated
CH3CN solution at -20 °C. The molecular geometries of 2a
and 2b are shown in Figs. 3 and 4, respectively. Consider-
ing the unit cell of 2a, there are eight asymmetry units in a
unit cell and there are four different molecules in every
asymmetry unit. Fig. 3 shows the geometries of these four
different molecules. The hydrogen’s of the acetonitrile can-
not be added because they exist on special positions but
they do not affect the quality of the structure data. Even
though these four molecules are different in 2a, there bond
lengths and angles are rather similar. All the four geome-
tries of 2a can be described as square pyramidal. The bond
lengths of Cu to oxygen atoms of methoxy fragments are
spanned in a large range from short-end 2.411 Å to long-
end 2.852 Å indicating the bonding to nonbonding interac-
tion of Cu to oxygen atoms. The average bond lengths of
Two types of green crystals of 2 were obtained from
CH3CN solution depending on the concentration and crys-
tallization time. Crystals of 2a were obtained directly from
Fig. 2. The molecular structure of compound 1. The at-
oms are drawn with 30% probability ellipsoids
and the hydrogen atoms are omitted for clarity.
698
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Cu(II) Compounds Effect on Colorectal Carcinoma Cell Growth
Table 2. The selected bond lengths (Å) and angles (°) for
compounds L1, 1 and 2 (2a and 2b)
center is best described as a distorted square based pyra-
mid. The four atoms constituting the basal plane are the two
nitrogen [N(1) and N(3)] atoms of the amine and pyrazole
and the two chlorine atoms [Cl(1) and Cl(2)] where the
bond angle of N(1)-Cu(1)-Cl(2) and N(3)-Cu(1)-Cl(1)
are 172.59(3)º and 170.10(4)º, respectively. The Cu(1)
atom is deviated from the square basal plane for ca. 0.15 Å.
The axial site is occupied by the O(2) atom with bond
length of Cu(1)-O(2) = 2.340(10) Å. In the basal plane the
average bond distances are Cu(1)-N(1), 2.133(12); Cu(1)-
N(3), 1.988(12); Cu(1)-Cl(1), 2.237(4) and Cu(1)-Cl(2),
2.261(4) Å.
L1
C(1)-N(1)
1.4425(13) C(13)-N(1)
1.4405(13)
C(17)-N(1)
1.4495(13) C(1)-N(1)-C(13) 118.13(8)
C(13)-N(1)-C(17) 118.00(9) C(1)-N(1)-C(17) 116.73(8)
N(1)-C(13)-N(4) 113.96(9) N(1)-C(17)-N(2) 113.81(9)
1
Cu(1)-Cl(1)
Cu(1)-N(1)
2.212(11) Cu(1)-Cl(2)
1.981(3) Cu(1)-N(5)
2.221(11)
1.997(3)
N(1)-Cu(1) -N(5) 125.99(13) N(1)-Cu(1)–Cl(1) 100.66(10)
N(5)-Cu(1) -Cl(1) 98.36(10) N(1)-Cu(1)–Cl(2) 100.61(10)
N(5)-Cu(1) -Cl(2) 103.56(10) Cl(1)-Cu(1)–Cl(2) 131.23(5)
UV-vis and ERP spectra measurements for com-
pounds 1 and 2
2a
Cu(1)-Cl(1)
Cu(1)-N(1)
Cu(2)-Cl(3)
Cu(2)-N(4)
Cu(2)-O(3)
N(1)-Cu(1)-N(3) 81.01(8)
N(3)-Cu(1)-Cl(2) 93.57(5)
2.273(7)
1.991(2)
2.261(7)
1.978(2)
2.372(19)
Cu(1)-Cl(2)
Cu(1)-N(3)
Cu(2)-Cl(4)
Cu(2)-N(6)
2.244(6)
2.123(2)
2.246(7)
2.141(2)
The UV-vis spectral data for 1 and 2, were recorded in
methylene chloride and acetonitrile, respectively within a
range of 200-1000 nm. The observed UV absorption bands
exhibit charge-transfer transitions (CT)16 in the range 217
N(1)-Cu(1)-Cl(2) 172.70(6)
N(1)-Cu(1)-Cl(1) 91.15(6)
N(3)-Cu(1)-Cl(1) 171.54(6) Cl(2)-Cu(1)-Cl(1) 93.96(3)
N(4)-Cu(2)-N(6) 80.06(8)
N(6)-Cu(2)-Cl(4) 93.51(6)
N(4)-Cu(2)-Cl(4) 164.45(7)
N(4)-Cu(2)-Cl(3) 91.79(6)
N(6)-Cu(2)-Cl(3) 170.10(6) Cl(4)-Cu(2)-Cl(3) 95.75(3)
N(4)-Cu(2)-O(3) 100.31(8) N(6)-Cu(2)-O(3) 76.82(8)
Cl(4)-Cu(2)-O(3) 91.87(5)
Cl(3)-Cu(2)-O(3) 99.34(6)
2b
Cu(1)-Cl(1)
Cu(1)-N(3)
Cu(1)-O(2)
N(1)-Cu(1)-N(3) 81.62(5)
N(1)-Cu(1)-Cl(1) 92.55(3)
2.237(4)
1.988(12) Cu(1)-N(1)
2.340(10)
Cu(1)-Cl(2)
2.261(4)
2.133(12)
N(3)-Cu(1)-Cl(1) 170.10(4)
N(3)-Cu(1)-Cl(2) 91.15(4)
N(1)-Cu(1)-Cl(2) 172.59(3) Cl(1)-Cu(1)-Cl(2) 94.84(14)
N(3)-Cu(1)-O(2) 89.07(4)
Cl(1)-Cu(1)-O(2) 97.83(3)
N(1)-Cu(1)-O(2) 79.97(4)
Cl(1)-Cu(1)-O(2) 98.35(3)
Fig. 3. The molecular structure of compound 2a. The
atoms are drawn with 30% probability ellip-
soids and the acetonitrile and hydrogen atoms
are omitted for clarity.
Cu to pyrazole-N atom (1.986 Å), is shorter than the corre-
sponding value involving the amine-N atom (2.132 Å). The
Cu-N(pyrazole) bond lengths are comparable with other
previously reported Cu-pyrazole complexes, [1.966(5) and
1.985(2) Å],12 [1.944(4), 1.954(4) and 1.942(4) Å],13
[1.933(2) and 2.013(2) Å]14 and [1.951(4) and 1.955(5)
Å].15 The bond lengths of Cu to chlorine atoms are ranged
from 2.244 to 2.273 Å.
The compound 2b (Fig. 4) consists of a five-coordi-
nated copper center that achieves a square pyramidal ge-
ometry on coordination from a tetradentate ligand, binding
through its NNO donor set like tridentate fashion and two
adjacent cis chlorine atoms. The geometry of the Cu(1)
Fig. 4. The molecular structure of compound 2b. The
atoms are drawn with 30% probability ellip-
soids and the hydrogen atoms are omitted for
clarity.
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Table 3. Effects of compounds 1 and 2 on the cell viability of
and 281 nm for 1, and 233 and 298 nm for 2. The UV ab-
sorption band observed at 395 and 399 nm for 1 and 2, re-
spectively, is attributed to the ligand-to-Cu(II) charge-
transfer transition (LMCT).16 The EPR spectra of pow-
dered compounds 1 and 2 at 77 K are shown in Fig. 5. Com-
pounds 1 and 2 show a g value at 2.75 and 2.06, respec-
tively, which are typical for isolated Cu(II) compounds
with one unpaired electron.
various human cancer cells
Untreated control
100.0 ± 4.80
Treated with compound
Cell line
1
2
A549 cell
85.8 ± 2.2
53.7 ± 4.7
COLO 205 cell
HT-29 cell
Hep3B cell
HepG2 cell
Huh7 cell
100.0 ± 10.5
100.0 ± 4.30
100.0 ± 4.80
100.0 ± 6.90
100.0 ± 4.90
100.0 ± 3.50
099.5 ± 14.2 19.0 ± 1.5
110.4 ± 15.2 38.9 ± 4.1
67.4 ± 5.1
77.5 ± 2.7
113.2 ± 5.60 101.8 ± 4.90
119.4 ± 12.1 62.0 ± 8.5
101.6 ± 3.20 54.8 ± 2.8
Magnetic Measurements of 2
The magnetic moment of compound 2 was obtained
by SQUID magnetometry with 10,000 Gausses of external
magnetic field over the temperature range 5-350 K, as
PLC5 cell
Cells were treated with 50 mM compounds 1 and 2 for 48 h. The
reported values are the means ± SD (n = 3).
shown in Fig. 6. The effective magnetic moment (meff
~
1.78 B.M) is quite stable in the temperature range 5-350 K,
which is close to the spin only value, meff = 1.73 B.M., ex-
pected for the S = 1/2 spin state of d9 Cu(II) species.
Effects of Compounds 1 and 2 on Cell Viability of
Human Cancer Cells
treated with 50 mM compounds 1 and 2 for 48 h. Table 3
shows that compound 2 had a strong inhibitory effect on
cell growth in human colorectal carcinoma cells (COLO
205 cells and HT-29 cells). The cell viability values of com-
pound 2 on COLO 205 cells and HT-29 cells were 19.0%
and 38.9%, respectively. Marín-Hernández et al.11 indi-
cated that some mixed chelate copper-based compounds
have been tested for anticancer activity. We would further
investigate the anticancer effects of compound 2 on intrin-
sically and extrinsically mediated pathways in human
colorectal carcinoma cells.
The inhibitory effects of compounds 1 and 2 on the
cell population growth of human lung carcinoma cells
(A549 cells), human colorectal carcinoma cells (COLO
205 cells and HT-29 cells), human hepatoblastoma cell
(Hep3B cells and HepG2 cells), and human hepatocellular
carcinoma cells (Huh7 cells and PLC5 cells) were deter-
mined by MTT assays. Various human cancer cells were
CONCLUSION
In this article, we have demonstrated the coordination
behavior of potentially neutral multi-dentate pyrazole
ligands [2,6-diisopropylphenyl]-bis[(1-H-pyrazol-1-yl)-
methyl]amine (L1) and pyrazole dimethoxethyl ligand
[(1-H-pyrazol-1-yl)methyl]-bis(2-methoxyethyl)amine
(L2) with copper chloride. Single crystal X-ray diffraction
analysis shows that 1 and 2 have tetra- or penta-coordina-
tion of the central copper atom. The results from an MTT
assay demonstrated that compound 2 had a strong inhibi-
tory effect on cell growth in human colorectal carcinoma
cells (COLO 205 cells and HT-29 cells). Future work will
explore the coordination mode and modification of the cur-
rent system using different substituents concerning the
pyrazole derivative to develop new generations of transi-
tion metal ions and characterize their activities as well as
the stereochemical control during the course of the reac-
tion.
Fig. 5. The X-band EPR spectra of compounds 1 (a)
and 2 (b) observed at 77 K using THF as sol-
vent.
EXPERIMENTAL SECTION
Fig. 6. Temperature dependence of the effective mag-
netic moments for a microcrystalline sample of
2 in the range of 5-350 K.
Materials and Physical Measurements
All the reactions were performed using standard
700
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Cu(II) Compounds Effect on Colorectal Carcinoma Cell Growth
Schlenk techniques under an atmosphere of high purity ni-
trogen or in glove box. CuCl2 (Aldrich), 2,6-diisopropylan-
iline, bis(2-methoxyethyl)amine, formaldehyde and pyra-
zole (Strem Chemical) were obtained commercially and
used as received. All solvents were distilled and stored in
solvent reservoirs which contained 4 Å molecular sieves
and were purged with nitrogen. 1H and 13C NMR spectra
were recorded on a Bruker Avance 300 spectrometer.
4H, NCH2CH2O), 3.13 (s, 6H, OMe), 3.29 (t, 4H,
NCH2CH2O), 4.91 (s, 2H, NCH2N), 6.03 (m, 1H, pyrazole
CH), 7.29 (m, 1H, pyrazole CH), 7.35 (m, 1H, pyrazole
CH). 13C NMR (CDCl3, ppm): d 51.3 (t, JCH = 133 Hz,
NCH2CH2O), 58.2 (q, JCH = 141 Hz, OMe), 69.4 (t, JCH
=
151 Hz, NCH2N), 70.9 (t, JCH = 141 Hz, NCH2CH2O),
104.6 (d, JCH = 175 Hz, pyrazole CH), 129.5 (d, JCH = 190
Hz, pyrazole CH), 138.5 (d, JCH = 184 Hz, pyrazole CH).
Anal. Calc. for C10H19N3O2 : C, 56.32; H, 8.98; N, 19.70.
Found, C, 56.11; H, 9.31; N, 19.80%.
1
Chemical shifts for H and 13C spectra were recorded in
ppm relative to the residual protons of CDCl3 (7.24, 77.0).
Elemental analyses were performed on a Heraeus CHN-OS
Rapid Elemental Analyzer at the Instrument Center of the
NCHU. EPR spectra were recorded on a Bruker EMS spec-
trometer using X-band frequency and UV-vis spectra were
measured on a Agilent 8453 spectrometer.
Synthesis of L1CuCl2 (1)
A 50 mL Schlenk flask charged wtih L1 (0.251 g,
0.744 mmol) and 10 mL of methylene chloride. The solu-
tion was added to a 10 mL methylene chloride solution of
anhydrous CuCl2 (0.1 g, 0.744 mmol) at 0 °C. The resulting
solution was stirred for 3 h and filtered through celite. The
resulting yellowish solid was recrystallized from a CH3CN
solution to generate 0.223 g of yellow crystals. Yield,
63.5%. Anal. Calc. for C20H27N5CuCl2: C, 50.90; H, 5.77;
N, 14.84. Found, C, 51.09; H, 5.88; N, 15.04%.
Synthesis of Ligands and Metal Complexes
Synthesis of Ligand, [2,6-diisopropylphenyl]-bis[(1-H-
pyrazol-1-yl)methyl]amine (L1)
A250 mLSchlenk flask charged with 2,6-diisopropyl-
aniline (24.44 g, 0.127 mol) was cooled to 0 °C and was
added 37% formaldehyde (20.57 g, 0.254 mol). The result-
ing solution was added dropwise a ethanol solution (70
mL) of pyrazole (17.62 g, 0.254 mol) and stirred for 12 h.
The volatiles were removed under vacuum at 70 °C and the
resulting solid was recrystallized from a toluene solution to
yield white crystals of product. Yield, 30.23 g (70.6%). 1H
NMR (CDCl3): 1.01 (d, 12H, CH3), 2.69 (m, 2H, PhCHMe2),
5.49 (s, 4H, NCH2N), 6.19 (m, 2H, pyrazole CH), 7.05-
7.18 (m, 3H, phenyl CH), 7.27 (m, 2H, pyrazole CH), 7.53
(m, 2H, pyrazole CH). 13C{1H} NMR (CDCl3): 24.5 (q, JCH
= 126 Hz, CH3), 27.9 (d, JCH = 130 Hz, PhCHMe2), 69.6 (t,
JCH = 150 Hz, NCH2N), 105.6 (d, JCH = 176 Hz, pyrazole
Synthesis of L2CuCl2 (2)
A 50 mL Schlenk flask was charged with L2 (1.586 g,
7.44 mmol) in 15 mL CH3CN. The solution was added
dropwise to a 15 mL CH3CN solution of anhydrous CuCl2
(1.0 g, 7.44 mmol) at 0 °C. The solution was stirred for 3 h
at room temperature and filtered through Celite to remove
unreacted CuCl2. The green filtrate was concentrated to
small amount and stored at -20 °C to afford 2.42 g of green
crystals of 2. Yield, 94%. Anal. Calc. for C10H19CuN3O2Cl2
: C, 34.54; H, 5.51; N, 12.08. Found, C, 34.25; H, 5.46; N,
11.99%.
X-ray Crystallography
CH), 124.3 (d, JCH = 156 Hz, phenyl CH), 127.6 (d, JCH
=
Good diffraction quality air stable single crystals of
L1, 1 and 2 were mounted on a Bruker SMART CCD
diffractometer equipped with graphite-monochromated
Mo-Ka radiation (l = 0.71073 Å). Crystal data were col-
lected at 150 K with an Oxford Cryosystems Cryostream.
No significant crystal decay was found. Data were cor-
rected for absorption empirically by means of y scans. All
non-hydrogen atoms were refined with anisotropic dis-
placement parameters. For both the structures, the hydro-
gen atom positions were calculated and they were con-
strained to idealized geometries and treated as riding where
the H atom displacement parameter was calculated from
the equivalent isotropic displacement parameter of the
bound atom. Data were processed using the CRYSALIS-
CCD and -RED programs.17 The structures of both com-
159 Hz, phenyl CH), 129.0 (d, JCH = 186 Hz, pyrazole CH),
139.7 (d, JCH = 185 Hz, pyrazole CH), 140.6 (s, phenyl
Cipso), 148.0 (s, phenyl Cipso). Anal. Calc. for C20H27N5: C,
71.18; H, 8.06; N, 20.75. Found; C, 70.89; H, 7.82; N,
20.28%.
Synthesis of Ligand [(1-H-pyrazol-1-yl)methyl]-bis(2-
methoxyethyl)amine (L2)
10 mL ethanolic solution of pyrazole (5.0 g, 72.0
mmol) was added to a mixture of bis(2-methoxyethyl)-
amine (9.68 g, 72.0 mmol) and formaldehyde (5.84 g, 72.0
mmol) in ethanol (50 mL) at 0 °C. The mixture was stirred
for 24 h at room temperature. The solvent was then evapo-
rated under vacuum to obtain the pure pale yellow liquid
L2. Yield, 14.75 g (96%). 1H NMR (CDCl3, ppm): d 2.62 (t,
J. Chin. Chem. Soc. 2012, 59, 696-703
© 2012 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jccs.wiley-vch.de
701

Article
Li et al.
plexes were determined by direct methods procedures in
SHELXS,18 and refined by full-matrix least-squares meth-
ods, on F2’s, in SHELXL.19
able microplate reader (Molecular Devices, Sunnyvale,
CA, USA) at 570 nm. The relative cell viability (%) related
to control wells containing cell culture medium without
samples was calculated by A570 nm [sample]/A570 nm [con-
trol] ´ 100.
Cell Culture
Human lung carcinoma cells (A549 cells) and human
hepatoblastoma cell (Hep3B cells and HepG2 cells) were
obtained from the Bioresource Collection and Research
Center (BCRC, Food Industry Research and Development
Institute, Hsinchu, Taiwan). Human hepatocellular carci-
noma cells (Huh7 cells and PLC5 cells) were obtained from
the American Type Culture Collection (ATCC, Bethesda,
MD, USA). Human colorectal carcinoma cells (COLO 205
cells and HT-29 cells) were provided by Dr Min-Hsiung
Pan (National Kaohsiung Marine University, Kaohsiung,
Taiwan). A549 cells were grown in a medium consisting of
90% RPMI 1640 with 10% fetal bovine serum supple-
mented with 0.1 mM nonessential amino acid, 2 mM L-
glutamine, 1 mM sodium pyruvate, and 100 units/mL peni-
cillin/streptomycin. COLO 205 cells and HT-29 cells were
grown in 90% RPMI 1640 medium supplemented with
10% fetal bovine serum, 100 units/mL penicillin, and 100
mg/mL streptomycin. Hep3B cells, HepG2 cells, Huh7
cells, and PLC5 cells were grown in 90% Dulbecco’s modi-
fied Eagle’s medium supplemented with 10% fetal bovine
serum, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1
mM non-essential amino acids, 1.0 mM sodium pyruvate,
100 units/mL penicillin, and 100 mg/mL streptomycin. Hu-
man cancer cells were cultured at 37 °C in a humidified 5%
CO2 incubator. Human cancer cells were treated with 50
mM compounds 1 and 2 for 48 h.
ACKNOWLEDGMENT
We acknowledge the financial assistance to the Na-
tional Science Council of Taiwan. We would also like to
thank the National Changhua University of Education for
supporting the X-ray diffractometer and NMR spectrome-
ter.
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