Metal-based biologically active agents: Synthesis, characterization, antibacterial and antileukemia activity evaluation of Cu(II), V(IV) and Ni(II) complexes with antipyrine-derived compounds

Article abstract of DOI:10.1016/j.ejmech.2009.10.034

The paper presents the synthesis of complex combinations of Cu(II), V(IV) and Ni(II) with Schiff bases obtained through the condensation of 4-amino-1,5-dimethyl-2-phenyl-1H-3-pyrazol-3(2H)-one (antipyrine) with 2-hydroxybenzaldehyde, 4-hydroxy-5-methoxyis

Full text of DOI:10.1016/j.ejmech.2009.10.034

European Journal of Medicinal Chemistry 45 (2010) 774–781
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Metal-based biologically active agents: Synthesis, characterization, antibacterial
and antileukemia activity evaluation of Cu(II), V(IV) and Ni(II) complexes with
antipyrine-derived compounds
,
a
a
a
c
b
Tudor Rosu a , Maria Negoiu , Simona Pasculescu , Elena Pahontu , Donald Poirier , Aurelian Gulea
*
a Inorganic Chemistry Department, Faculty of Chemistry, University of Bucharest, 23 Dumbrava Rosie Street, 020462 Bucharest, Romania
b Coordination Chemistry Department, Moldova State University, 60 Mateevici Street, Kishinev, Moldavia
c Oncology and Molecular Endocrinology Research Center CHUL, Research Center and Universite Laval, CHUQ-CHUL, 2705 Boulevard Laurier, Quebec City G1V4G2 Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
The paper presents the synthesis of complex combinations of Cu(II), V(IV) and Ni(II) with Schiff bases
obtained through the condensation of 4-amino-1,5-dimethyl-2-phenyl-1H-3-pyrazol-3(2H)-one (anti-
pyrine) with 2-hydroxybenzaldehyde, 4-hydroxy-5-methoxyisophthalaldehyde and 4,5-dihydroxy
Received 14 July 2009
Received in revised form
22 October 2009
Accepted 23 October 2009
Available online 4 November 2009
isophalaldehyde respectively. The characterization of newly formed complexes was done by 1H NMR, 13
C
NMR, UV–VIS, IR, EPR spectroscopies and molar electric conductibility studies. The effect of these
complexes on proliferation of human leukemia cells (HL-60) and their antibacterial activity against
Staphylococcus aureus var. Oxford 6538, Escherichia coli ATCC 10536 and Candida albicans ATCC
10231strains were studied and compared with those of free ligands.
Keywords:
Schiff bases
Antimicrobial activity
Leukemia cells HL-60
Copper complexes
Antipyrine
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
This paper is a continuation of our previous research and it
presents the synthesis and characterization of new complexes of
Schiff base complexes are considered to be among the most
importantstereochemicalmodels in main group andtransition metal
coordination chemistry due to their preparative accessibility and
structural variety [1]. Schiff bases are potential anticancer drugs and,
when administered as their metal complexes, the anticancer activity
of these complexes is enhanced in comparison to the free ligand [2].
Schiff bases of 4-aminoantipyrine and its complexes present
a great variety of biological activity ranging from antitumor,
fungicide, bactericide, anti-inflammatory, and antiviral activities
[3–5]. The number of transition metal complexes of Cu(II), Ni(II)
and V(IV) with oxygen and nitrogen donor Schiff base derivatives of
4-aminoantipyrine is limited. A small number of papers describe
the synthesis and characterization of these compounds based on
aminoantipyrine Schiff bases [6–14].
Cu(II) with Schiff base L1, using other salts of Cu(II) as well as other
complexes of V(IV) and Ni(II). Also, this paper presents the synthesis
and the characterization of some complexes of Cu(II) and V(IV) with
newly Schiff bases (Fig. 1) obtained by condensation of 4-amino-
antipyrine with 4-hydroxy-5-methoxyisophthalaldehyde, L2 and
4,5-dihydroxy-isophtalaldehyde L3, respectively. The biochemical
effects involved in the anti-proliferative activity of ligands and
synthesized complexes in human HL-60 promyelocytic leukemia
cells were investigated. The complexes and ligands were also tested
for their in vitro antibacterial activity against Staphylococcus aureus
var. Oxford 6538, Escherichia coli ATCC 10536 and Candida albicans
ATCC 10231 strains using the paper disc diffusion method [16] (for
the qualitative determination) and the serial dilutions in liquid
broth method [17] (for determination of MIC).
In a previous paper [15] we presented the synthesis of Cu(II)
complexes derived from the newly Schiff base ligands obtained by
condensation of 4-amino-antipyrine with 2-hydroxybenzaldehyde
L1 and terephthalic aldehyde L4 using CuCl2$2H2O and CuSO4$5H2O
salts, respectively.
2. Results and discussion
2.1. Chemistry
The present Schiff bases L2- L3 (Fig. 1) were prepared under the
method described elsewhere [15], by refluxing in methanol
* Corresponding author. Tel.: þ40 21 2103497.
E-mail address: t_rosu0101@yahoo.com (T. Rosu).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.034

T. Rosu et al. / European Journal of Medicinal Chemistry 45 (2010) 774–781
775
assignments. All the protons were found to be in the expected
regions [19]. It was also observed that DMSO did not have any
coordinating effect on the ligands or their metal complexes.
19
CH3
O
15
HO
16
14
2.1.2. Electronic spectra and magnetic moments
The geometry of the metal complexes has been deduced from
electronic spectra and magnetic moment data of the complexes
(Table 1). The complex [Cu2(C18H16N3O2)2]SO4 (1) shows a very low
magnetic moment measured at room temperature, 0.62 BM per
copper center. The subnormal magnetic moment indicates that the
copper centers are strongly coupled anti-ferromagnetic spin-spin
interactions through molecular association for square-planar
geometry (Fig. 2) [20–25], or indicates the fact that a super-
exchange interaction is probably occurs [26a]. The reflectance
spectra exhibit broad absorption band at 14710 cmꢁ1 specific for
most complexes of Cu(II) [26b] and a weak band at 20408 cmꢁ1. The
values of the magnetic moment (1.88–1.94 BM) of the solid
20
12
20'
H3C
12'
CH3
C
H
N
3
17
13
5
N
3'
C
4
18
4'
5'
H
1
21
CH3
1'
N
2
N
21'
2'
O
N
O
H3C
N
6
6'
7
11
10
7'
8'
11'
10'
8
(L2)
9
9'
complexes [Cu(C18H16N3O2)2] (2) and (3) as well as [Cu(C18H16
-
N3O2)(H2O)2]ClO4 (4) indicate the presence of an unpaired electron
on Cu(II) ion.
The reflectance spectra exhibit three absorption bands of
medium intensity for the complexes (2) and (3): 14320 cmꢁ1
,
OH
15640 cmꢁ1, and 18270 cmꢁ1, which may be attributed to the
15
HO
16
transitions dx2 / d2z, dx2 / dxz,yz and dx2 / dxy charac-
14
ꢁy2
ꢁy2
ꢁy2
teristic to a distorted octahedral geometry. The reflectance spectra
for complex (4) present a single absorption band at 14890 cmꢁ1
corresponding to the d–d transition, indicating the low C2V
symmetry of the Cu2þ ion [26c]. The magnetic moment value (1.78
BM) of the complex [Cu(C18H16N3O2)2(H2O)2(ac)2] (5) indicates
that a super-exchange interaction is occurring, probably ps / es or
pp / t2g [26d,e]. The reflectance spectra of this complex showed
two weak, low-energy bands at 13880 cmꢁ1 and 19050 cmꢁ1
19
12
C
19'
12'
CH3
N
3
17
13
H3C
5
N
3'
C
4
18
H
4'
5'
H
1
20
1'
N
2
20'
N
2'
O
CH3
N
O
H3C
N
6
6'
7
11
10
7'
8'
11'
10'
attributed to the transitions dx2 / dxz,yz and dx2 / dxy
ꢁy2
ꢁy2
8
specific to a distorted octahedral geometry and a strong high-
energy band at 26320 cmꢁ1, assigned to ligand/metal charge
transfer [27,28].
(L3)
9
9'
Also, at room temperature the magnetic moment values (2.02,
2.04 BM) of the solid complexes [Cu(C31H30N6O4)]SO4 (8) and
[Cu(C30H28N6O4)]SO4 (10) indicate the presence of an unpaired
electron on Cu(II) ion in an ideal square-planar environment [20].
The electronic spectra of complexes (8) and (10) showed two weak,
low-energy bands at 14980, 20870 cmꢁ1 and 15160, 21290 cmꢁ1
respectively, which may be assigned to 2B1g / 2A1g and 2B1g / 2Eg
transitions, for their square-planar geometry [29]. The magnetic
moment values (1.51–1.73 B.M) for the VOþ2 complexes
[VO(C18H16N3O2)(H2O)]2SO4 (6), [VO(C31H30N6O4)]SO4 (9) and
[VO(C30H28N6O4)]SO4 (11) suggest the presence of an unpaired
electron and a strongly distorted geometry. All these complexes are
paramagnetic and their values show the existence of monomeric
species of oxovanadium (IV) [29].
Fig. 1. The structures of ligands L2, L3.
(30–40 mL) an equimolar mixture of 4-amino-1,5-dimethyl-2-
phenyl-1H-3-pyrazol-3(2H)-one with 4-hydroxy-5-methoxy
isophthalaldehyde or 4,5-dihydroxyisophalaldehyde. The
structures of these formed Schiff bases were established by IR, UV,
1H NMR and 13C NMR spectroscopies. These Schiff bases were
further used for the complexation reaction with Cuþ2, VOþ2 and
Niþ2 metal ions, using the following metal salts: CuSO4$5H2O (for
complexes 1, 2, 8, 10), Cu(NO3)2 3H2O (for 3), Cu(ClO4)2$6H2O (for
4), Cu(OAc)2$H2O (for 5), NiCl2$6H2O (for 7) and VOSO4$2H2O
(for 6, 9, 11) (Merck). The obtained complexes are microcrystalline
solids which are stable in air and decompose above 250 ꢀC (Table 1)
(except for complexes 1, 5, 6). They are insoluble in organic solvents
such as acetone and chloroform, but soluble in methanol, DMF and
DMSO. The molar conductance of the soluble complexes in DMF
showed values indicating that (2), (3), (5) and (7)
(10–15 ohmꢁ1 cm2 molꢁ1) are non-electrolytes and (1), (4), (6),
(8–11) (80–90 ohmꢁ1 cm2 molꢁ1) are electrolytic in nature [18].
With the elemental analyses data of the Schiff bases (reported in
experimental) and their complexes (Table 1) are in agrement with
structures of the ligands as shown in Fig. 1 and with of the formulae
of the complexes as shown in Fig. 2.
The electronic spectra of complex (6) exhibit three bands. The
first band at z14250 cmꢁ1 can be attributed to a d-d transition
(dxy / dxz;yz.). The second band at z20250 cmꢁ1 can be assigned
to a dxy / d
transition. The third band at about 24520 cmꢁ1 is
22
xꢁy
ꢁy2
attributed to a dxy / dx2
transition. The electronic spectra of
complexes (9) and (11) exhibit two bands of low intensity at
13680, 18940 cmꢁ1 and 13180, 19120 cmꢁ1 respectively, due to
the ligand-field transitions dxy / dxz;yz. and dxy / dx2ꢁy2, both
characteristic for an oxovanadium(IV) chromophore. A third d-
d transition was probably obscured by the tail of the ligand-to-
metal or intra-ligand charge-transfer absorption [30]. The value of
meff (3.5 BM) obtained for complex [Ni(C18H16N3O2)2] (7) corre-
sponds to two unpaired electrons per Ni(II) ion for their six-
coordinated configuration [20]. The Ni(II) complex exhibited three
spin-allowed bands at 10360, 16220 and 28940 cmꢁ1 assignable to
2.1.1. NMR spectra
The NMR spectra of free ligands were determined in DMSO-d6.
The 1H NMR spectral data are reported along with the possible

Table 1
Physical and analytical data of the metal complexes (1-11) and ligands L1(*), L2 and L3
IR (cmꢁ1
)
n
e(l
lmax
(cmꢁ1
)
2-
Comp No
Molecular
formula
Molecular
mass
Yield
(%)
M.p.
(0C)
m
eff
Elemental analysis (%) calc (found)
SO4
,, nm)
(BM)
(cm- 1
)
C
H
N
M
L1
(1)
C18H16N3O2
[Cu2(L1)2]SO4
835.83
676.22
676.22
505.37
893.88
878.10
671.37
550.61
710.22
713.61
536.58
696.19
699.59
67
78
83
79
81
74
85
87
65
205-207
>250
0.62
51.73
(51.95)
63.94
(64.05)
63.94
(64.10)
42.78
(42.90)
53.75
(53.89)
49.21
(49.38)
64.40
(64.67)
67.62
(68.01)
52.43
(52.58)
52.18
(52.33)
67.15
(67.36)
51.76
3.82
(3.65)
4.73
(4.58)
4.73
(4.58)
3. 95
(3.72)
4.69
(4.47)
4.09
(3.83)
4.76
(4.52)
5.44
(5.27)
4.22
(4.05)
4.20
(3.98)
5. 21
(5.07)
4.02
(3.84)
4.00
(3.87)
10.05
(9.21)
12.43
(12.31)
12.43
(12.34)
8. 31
(8.14)
9.40
(9.31)
9.56
(9.32)
12.52
(12.31)
15.26
(15.11)
11.83
(11.65)
11.78
(11.59)
15.66
(15.32)
12.07
(11.88)
12.01
(11.88)
15.21
(15.03)
9.40
(9.28)
9.40
(9.26)
12.57
(12.38)
14.22
(14.13)
11.60
(11.43)
8.74
(8.59)
-
-
8. 95
(8.81)
7.14
(6.93)
-
1645(C¼O),1134(Ar-OH), 1585(C¼N)
1628(C¼O),1126(Ar-OH), 1585(C¼N)
1620(C¼O),1130(Ar-OH), 1584(C¼N)
1624(C¼O),1120(Ar-OH), 1582(C¼N)
1622(C¼O),1120(Ar-OH), 1598(C¼N)
1664(C¼O),1130(Ar-OH), 1584(C¼N)
1642(C¼O),1128(Ar-OH), 1581(C¼N)
1148
613
e490
15 ꢂ 103
e550
6.3 ꢂ 103
e550
6.8 ꢂ 103
e670
4.3 ꢂ 103
e525
10 ꢂ 103
e410
5 ꢂ 103
e345
13 ꢂ 103
14710; 20408
C36H32Cu2N6O8S
[Cu(L1)2]
C36H32CuN6O4
[Cu(L1)2]
C36H32CuN6O4
[Cu(L1)(H2O)2]ClO4
C18H20CuClN3O8
[Cu2(L1)2(H2O)2(OAc)2]
C40H42Cu2N6O10
[VO(L1)(H2O)]2SO4
(2)
(3)
(4)
(5)
(6)
(7)
L2
1.94
1.92
1.88
1.78
1.51
3.5
14320;15640;
18270
14320;15640;
18270
>250
>250
14890
238-240
201-202
>250
13880;19050
1145
610
14250; 20250; 24520
10360; 16220; 28940
C36H36V2N6O12
S
[Ni(L1)2]
C36H32NiN6O4
C31H30N6O4
153-154
>250
-
1664(C¼O),1134(Ar-OH),
1610(C¼N)
(8)
(9)
L3
[Cu(L2)]SO4
C31H30CuN6O8S
[VO(L2)]SO4
C31H30VN6O9S
C30H28 N6O4;
2.02
1.73
-
1623(C¼O),1130(Ar-OH),
1583(C¼N)
1145
1149
620
612
e480
16 ꢂ 103
e530
4.8 ꢂ 103
14980; 20870
13680; 18940
>250
1679(C¼O),1138(Ar-OH),
1590(C¼N)
80
59
53
199-200
>250
1655(C¼O),1138(Ar-OH),
1610(C¼N)
-
(10)
(11)
[Cu(L3)]SO4
C30H28CuN6O8S
[VO(L3)]SO4
2,04
1.60
9.13
1629(C¼O),1136(Ar-OH),
1579(C¼N)
1142
1147
604
609
e470
14 ꢂ 103
e525
4.8 ꢂ 103
15160; 21290
13180; 19120
(51.94)
51.50
(51.77)
(8.94)
7. 28
(7.09)
>250
1673(C¼O),1131(Ar-OH),
1578(C¼N)
C30H28VN6O9S
(*) L1 was prepared and described in [15]

T. Rosu et al. / European Journal of Medicinal Chemistry 45 (2010) 774–781
777
CH
H3C
C
O
CH
N
O
N
CH3
H2
H3C
N
O
N
O
O
H3C
N
Cu
O
O
2-
O
CH3
N
SO4
Cu
N
Cu
O
Cu
N
CH3
H3C
N
N
O
H3C
N
O
O
N
O
CH3
C
H2O
HC
HC
CH3
(1) [Cu2(L1)2](SO4)
[Cu2(L1)2(H2O)2(OAc)2]
(5)
+
CH
O
CH
CH3
N
N
CH3
H3C
O
N
O
V
N
H3C
M
O
O
N
CH3
2-
N
SO4
N
O
CH3
N
N
O
H2O
HC
2
(6) [VO(L1)(H2O)]2(SO4)
(2) [Cu(L1)2] -from CuSO4x5H2O
(3)
[Cu(L1)2] - from CuNO3x3H2O
(7) [Ni(L1)2]
X
HO
HC
N
CH
N
H3C
CH3
2-
HC
O
SO4
M
H3C
N
N
N
H3C
CH3
N
N
O
O
Cu
H2O
-
N
ClO4
O
O
H2
N
H3C
[Cu(L2)](SO4)
[VO(L2)](SO4)
, (8)
X=OCH3
(9)
[Cu(L1)(H2O)2](ClO4)
(4)
[Cu(L3)](SO4)
, (10)
X=OH
[VO(L3)](SO4)
(11)
Fig. 2. Proposed structures of the newly obtained metal complexes.
3
3
3A2g(F) / 3T2g(F) (y1), A2g(F) / 3T1g(F) (y2) and A2g(F) / 3T2g(P)
2.1.3. IR spectra and coordination mode
(
y3) transitions, which are characteristic for their octahedral
The most relevant bands in the infrared spectra of the
complexes are presented in Table 1. IR spectra of the ligands
showed the absence of the bands at z1740 cmꢁ1 and z3400 cmꢁ1
geometry [20,31]. For all the complexes was calculated molar
absorption (Table 1).

778
T. Rosu et al. / European Journal of Medicinal Chemistry 45 (2010) 774–781
due to carbonyl
y(C]O) and amino
y(NH2) stretching vibrations
between the Cu(II) centers in polycrystalline compounds following
and, instead, a new band assigned to azomethine
y
(CH]N) linkage
the formula G ¼ (g ꢁ 2)/(gt ꢁ 2) [28]. If G < 4, it is considered the
k
appeared at z1612 cmꢁ1 [32]. This suggested that amino and
carbonyl groups of the starting reagents (4-amino-antipyrine and
substituted benzaldehyde, respectively) have been converted into
their corresponding Schiff bases (Fig. 1).
existence of some exchange interactions between the Cu(II) centers
and if G > 4, the exchange interactions are neglected. Thus, in case
of complex (1), the geometric parameter G ¼ 3.68 confirms the
existence of some exchange interactions between the Cu(II)
centers. For the complexes (2), (3) and (8), the geometric parameter
G is however higher than 4.
Bands at 1614–1610 cmꢁ1 due to C]N stretching frequencies in
the free ligands shift towards lower values (1590–1578 cmꢁ1) in all
complexes indicating that the azomethine nitrogen atom was
coordinated. Bands at 1664–1653 cmꢁ1 due to C]O stretching
frequencies of antipyrine moiety, in the free ligands shift towards
lower values in the spectra of the complexes of the Cuþ2 and Niþ2
The g tensor values for the complex (4) indicate a distorted
trigonal bipyramide geometry (C2v) [41]. The term of the funda-
mental state may be defined with the help of the geometric
parameter R deduced from the relation R ¼ (g2 ꢁ g3)/(g1 ꢁ g2) [36].
If R > 1, the term of the ground state is defined by the orbital d2z, but
if R < 1 it is defined by the orbital dx2ꢁy2. For the complex (4)
(R ¼ 2.46), it is clear that the ground state term is d2z. From the value
of the R parameter for complex (5), the term of the ground state is
ions and towards higher values in all complexes of VOþ2
Comparing the spectra of Cuþ2 ion complexes with the ones of
VOþ2, be noticed that the (C]O) frequencies of the VOþ2
.
y
complexes spectra are moving by 11–18 cmꢁ1 in a positive way as
compared to the same frequencies of the ligands. This displacement
can be attributed to the electronic donation of the base to the
vanadium (N / V), which increases the electron density on the
metal d-orbitals, and consequently the pp / dp donation from
the oxygen atom to vanadium is expected to be reduced [30]. This
behavior indicates the fact that the carbonyl oxygen atom of the
antipyrine residue was coordinated. The IR spectra of complex (5)
exhibit two bands at 1580 and 1425 cmꢁ1 which are characteristic
for nasym (COOꢁ) and nsym(COOꢁ) of the symmetrical group COOꢁ
present in bridging complexes [32].
ꢁy2
defined by dx2
orbital. This result is in agreement with the data
obtained by electronic spectra. From the g tensor values for the
complex (10), there was determined the value of the parameter R
ꢁy2
indicating a term of the ground state defined by dx2
orbital.
2.2. Biological activity
2.2.1. Cell viability determination
The in vitro cytotoxicity of the ligands L2, L3 and some of their
Cu(II), Ni(II) and VO(IV) complexes (1), (6), (7), (8), (9), (10) and (11)
on human promyelocytic leukemia cells (HL-60) was determined
by a MTS-based assay. Included in this section are values for 4-(2-
hydroxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one L1, 4,4’-(1,4-phenylene bis(iminomethyl)-bis(1,5-
dimethyl-2-phenyl-1H-pyrazol-3(2H)-one), L4 and copper(II)
complex [Cu2(L4)2(SO4)2] (12), previously obtained [15]. The cells
were incubated 3 days in the presence of each ligand or complex at
Also, the specific band of aromatic-OH (1140 cmꢁ1) from the free
ligand L1 [15] moves towards smaller wave numbers in the IR
spectra of the complex combinations (1–7) (1120–1134 cmꢁ1). This
effect always indicates the coordination of the ligand through the
oxygen atom of the OH phenolic group.
The IR spectra of the VOþ2 complexes exhibit a very strong band
at 990–975 cmꢁ1 attributed to the stretching vibration of the
terminal V]O bond [33,34]. Based on these observations it may be
appreciated that the L1 coordinate tridentate mononegatively
around the metallic ion and the L2 and L3 coordinate neutral
tetradentately.
two concentrations (1 and 10 mM) and the results were expressed
as the percentage of cell growth (Table 3). The cell proliferation of
the control (untreated cells) was fixed to 100%. Any tested ligands
and complexes inhibited the cell proliferation at a concentration of
1
10
m
M, but complexes (8), (10) and (12) shown a significant effect at
2.1.4. EPR spectroscopy
mM. The copper(II) complex (12) was clearly the most potent
EPR spectra of the polycrystalline powders were recorded at
room temperature. The values of g factors were assessed following
the method described by J. W. Searl [35] and are presented in
Table 2. The g tensor values of Cu(II) complex can be used to derive
the ground state. In tetragonal and square planar complexes the
unpaired electron lies in the dx2
state with the g//> gt > 2.003 [36–40]. From the observed values
for complexes (1-3) and (8), it is clear that g > gt. These data are in
agreement with those obtained from the electronic spectra and
confirm the tetragonal geometry for complexes (2) and (3) and the
square planar geometry for complexes (1) and (8). The term of
the fundamental state is thus defined by the orbital dx2ꢁy2. From the
values of the g factors it may be determined the geometric
parameter G, representing a measure of the exchange interaction
compound reducing the HL-60 cell proliferation to 40% whereas
complexes (8) and (10) reduced the cell proliferation to 82% and
orbital giving 2B1g as the ground
Table 3
ꢁy2
Effect of a series of ligands L1–L4, and complexes (1), (6–12) at two concentrations of
1 and 10 m
M on the HL-60 cell proliferation.a
k
Compound
Survival
cell fraction (%)
Cell growth
inhibition (%)
Survival cell
fraction (%)
Cell growth
inhibition (%)
at 1
m
M
at 1
m
M
at 10
m
M
at 10 mM
CTL
L1
(1)
100 ꢃ 2
110 ꢃ 3
119 ꢃ 2
112 ꢃ 1
116 ꢃ 2
119 ꢃ 4
90 ꢃ 1
–
–
–
–
–
–
100 ꢃ 2
113 ꢃ 2
94 ꢃ 2
110 ꢃ 2
98 ꢃ 1
94 ꢃ 3
81 ꢃ 4
110 ꢃ 2
101 ꢃ 4
69 ꢃ 3
145 ꢃ 5
111 ꢃ 1
41 ꢃ 2
5 ꢃ 1
–
–
6
(6)
–
(7)
2
6
L2
(8)
10b
–
–
–
–
–
19b
–
(9)
110 ꢃ 1
99 ꢃ 2
Table 2
L3
–
The {g} parameter values of the Cu(II) complexes.
(10)
(11)
L4
103 ꢃ 4
114 ꢃ 2b
115 ꢃ 3
104 ꢃ 4
20 ꢃ 2
31b
–
b
Nr
Complex
g
k
gt
g1
g2
g3
–
(1)
(2)
(3)
(4)
(5)
(8)
[Cu2(C18H16N3O2)2]SO4
[Cu(C18H16N3O2)2]
[Cu(C18H16N3O2)2]
[Cu(C18H16N3O2)(H2O)2]ClO4
[Cu2(C18H16N3O2)2(H2O)2(OAc)2]
[Cu(C31H30N6O4)]SO4
2.247 2.067
2.245 2.052
2.243 2.060
(12)
–
59b
95b
Doxoc
80b
2.263 2.209 2.076
2.207 2.107 2.058
a
The results ꢃ SD are reported as the survival fraction of cells (%). Cell growth
inhibition in % was also reported for active compounds.
2.268 2.064
b
Significantly different from the control (P > 95%).
The potent cytotoxic agent doxorubicin (Doxo) was used as a positive control.
(10) [Cu(C30H28N6O4)]SO4
2.461 2.178 2.066
c

T. Rosu et al. / European Journal of Medicinal Chemistry 45 (2010) 774–781
779
70%, respectively. Since no effect was observed with the ligand (L2,
L3 and L4) only, the inhibition of malignant cell proliferation
observed with (8), (10) and (12) is the result of a copper-ligand
complexation. On the other hand, the vanadium complexes (9) and
(11), which are analogues to the copper complexes (8) and (10), did
not reduce the cell proliferation. In fact, the vanadium complex (11)
stimulated the cell proliferation of HL-60 cells.
the study. Compound (3), was found to have low activity against
S. Aureus, E. coli and C. albicans (MIC ¼ 512 g/mL) probably due to
the presence of more bulkier ClOꢁ4 anion. The most active tested
compounds are (9) and (11), (MIC ¼ 64–128 g/mL), probably due
to the simultaneous presence of VO2þ and SO42ꢁ
The present investigations of antimicrobial screening data
revealed that all of the newly synthesized compounds exhibited
poor activity compared to that of the control drugs. Because the
MIC values are not spectacular, no statistical calculations were
made.
m
m
.
2.2.2. Antibacterial activity
A comparative study of MIC values of the Schiff bases L2-L3 and
its complexes indicate that, generally, the metal complexes have
a better activity than the free ligands. This is probably due to the
greater lipophilic nature of the complexes. Such increased activity
of the metal chelates can be explained on the basis of chelating
theory [35]. On chelating, the polarity of the metal ion will be
reduced to a greater extent due to the overlap of the ligand orbital
and partial sharing of positive charge of the metal ion with donor
3. Conclusions
The analytical and physico-chemical analyses confirmed the
composition and the structure of the newly obtained complex
combinations. The IR, electronic transition and {g} tensor value data
lead to the conclusion that the Cu2þ ion takes different geometries
depending on the nature of used metallic salts, solvent (complexes
(1) and (2)) and ligand type. In all complexes the ligand L1 acts as
mononegative tridentate, around the metallic ion, and the ligands
L2 and L3 coordinate neutral tetradentate.
Results of antitumor activity screening indicated that the ligand
only has no cytotoxic effects at the two tested concentrations.
Interestingly, the complex (12) greatly reduced the malignant HL-
60 cell growth. The sensitivity spectrum of the microbial strains
towards the ligands and the corresponding complexes was deter-
mined by qualitative and quantitative methods. The quantitative
antimicrobial activity test results proved that both the ligand and
the complex combinations have specific anti-microbial activity,
depending on the microbial species tested. The antibacterial assay
against other Gram-negative and Gram-positive strains is in prog-
ress, because none of the presented compounds is effective against
the tested micro-organisms in comparison with used drugs.
groups. Further, it increases the delocalization of
p-electrons over
the whole chelate ring and enhances the lipophilicity of the
complex. This increased lipophilicity enhances the penetration of
the complexes into lipid membrane and blocks the metal binding
sites on enzymes of micro-organisms.
The synthesized Schiff base compounds have comparable and
similar inhibitory effects (low to moderate MIC values 256 and
512 mg/mL) on the growth of tested strains (Table 4). The antibac-
terial results evidently show that the activity of the Schiff base
compounds became more pronounced when coordinated to the
metal ions. Hence, all the complexes show greater bactericidal and
fungicidal activities against E. coli (MIC ¼ 128
mg/mL) and C .albicans
g/mL) as compared to their corresponding Schiff bases
(MIC ¼ 128
m
(exception for (7), which exert a visible decrease of antimicrobial
action).
The structure of the tested compounds seems to be the principal
factor influencing the antimicrobial activity. The presence of
anionic groups outside the coordination site, exerts a number of
changes on antimicrobial activity of the tested complexes. So, in
case of (1), (4), (8–11), the presence of SO24ꢁ moiety induced
a visible increase of their action against all bacterial species taken in
4. Experimental protocols
4.1. Chemistry
The required chemicals were of analytical reagent grade and
were purchased from Merck and Chimopar Bucharest. All manip-
ulations were performed using materials as received. Electronic
spectra were recorded using the Jasco V-550 spectrophotometer, in
diffuse reflectance, using MgO dilution matrices. IR spectra were
recorded with a BioRad FTS 135 spectrophotometer in the 4000–
400 cmꢁ1 region using KBr pellets. EPR spectra were recorded on an
ART-6 a spectrometer, equipped with a field modulation unit at
100 kHz. Measurements were effected in the X band, on micro-
crystalline powder at room temperature. The 1H-NMR and 13C-
NMR spectra were recorded in CDCl3, with the Bruker DRX 400
spectrometer. The molar conductivity was determined with
CONSORT-C533 conductometer. The chemical elemental analysis
for the determination of C and N was done with the Carlo-Erba LA-
118 microdosimeter whereas the AAS-1N Carl-Zeiss-Jena spec-
trometer was used for the determination of Cu(II) and Ni(II).
Vanadium was determined following the method described by
Fries J. and Getrost H. [42].
Table 4
Antibacterial activities of ligands L1–L3 and complexes (1–11) as MIC values (
mg/mL).
Compound
Gram-negative bacteriaa
Sa Ec
512 256
Fungb
Ca
L1
(1)
(2)
(3)
(4)
(5)
(6)
512
128
512
512
128
128
128
1024
128
128
128
128
128
64
512
–
1024
–
1024
512
–
256
512
512
256
512
256
512
512
128
256
512
256
128
1024
1024
–
128
1024
512
128
128
128
1024
256
128
128
–
(7)
L2
(8)
(9)
L3
(10)
(11)
128
64
CuSO4 5H2O
Cu(NO3)2 3H2O
Cu(ClO4)2 6H2O
Cu(OAc)2 H2O
NiCl2 6H2O
VOSO4 2H2O
Tetracycline
Fluconazol
512
1024
1024
512
–
4.1.1. General procedures for the synthesis of the Schiff bases L1, L2
and L3
512
–
L1: 4-(2-hydroxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-
pyrazol-3(2H)-one has been prepared by the method described
elsewhere [15].
512
1024
0.19
1.5
L2:
4,40-(4-hydroxy-5-methoxy-1,3-phenylenebis(iminomethyl)-
–
–
2
a
bis(1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one). A solution of
4-hydroxy-5-methoxy-isophthalaldehyde (0.018 g, 1 mmol) in
Sa (Staphylococcus aureus var. Oxford 6538); Ec (Escherichia coli ATCC 10536).
Ca (Candida albicans ATCC 10231).
b

780
T. Rosu et al. / European Journal of Medicinal Chemistry 45 (2010) 774–781
methanol (10 mL) was added to a solution of 4-amino-1,5-dimethyl-
2-phenyl-1H-pyrazol-3(2H)-one (0.0203 g, 1 mmol) in methanol
(20 mL). The mixture was refluxed for 1 h, then stirred for 3 h at
room temperature and left at the same temperature for one day. The
resulting precipitate of intense yellow color was filtered, washed
with methanol and dried.
(Sigma, Saint Louis, USA) containing L-glutamine (2 nM), antibiotics
(100 IU penicillin/mL, 100 g streptomycin/mL) and supplemented
with 10% (v/v) fetal bovine serum (FBS), in a 5% CO2 humidified
atmosphere at 37 ꢀC. Cells were currently maintained in continuous
exponential growth with twice a week dilution of the cells in
culture medium.
m
Yield: 87%; M.p. 153-154 ꢀC. Anal. Calc. (%) for C31H30N6O4
(550.61 g/mol): C-67.62; N-15.26; Found: C-68.01; N-15.11; IR (KBr,
cmꢁ1): The IR spectrum of the obtained ligand confirms the
occurrence of the absorption band of 1610 cmꢁ1, specific for the
azomethinic group [26,27]; 1664 (s, C]O), 1144 (Ar-OH); 1H-NMR
4.2.3. Cell proliferation assay
The cell proliferation assay was performed using 3-(4,5-
dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium inner salt (MTS) (Cell Titer 96 Aqueous,
Promega, USA), which allowed us to measure the number of
viable cells. In brief, triplicate cultures of 1 ꢂ104 cells in a total of
(DMSO-d6, d
, ppm): 2.16 (s; 3H; H-20); 2.42 (s; 3H; H-200); 3.15 (s;
3H; H-21); 3.22 (s; 3H; H-210); 3.89 (s; 3H; H-19); 7.35-7.57 (m;
10H; H-7-11; H-70-110); 7.80 (s; 1H; H-14); 7.86 (s; 1H; H-18); 9.50
(s; 1H; H-12); 9.73 (s; 1H; H-120).
100 mL medium in 96-well microtitre plates (Becton Dickinson
and Company, Lincoln Park, NJ, USA) were incubated at 37 ꢀC, 5%
CO2. Compounds were dissolved in DMSO to prepare the stock
solution of 1 ꢂ10ꢁ2 M. These compounds were diluted at the
13C-NMR (DMSO-d6;
d, ppm):163.7 (C-3; C-3’); 160.5 (C-12; C-
120); 154.2 (C-16); 153.6 (C-15); 150.3 (C-5; C-50); 133.9 (C-6; C-
60); 129.2 (C-8; C-10; C-80; C-100); 127.4 (C-13); 123.9 (C-7; C-11;
C-70; C-110); 122.8 (C-9; C-90); 122.1 (C-18); 119.6 (C-17); 113.5 (C-
14); 111.1 (C-4; C-40); 55.8 (C-19); 34.5 (C-210); 33.9 (C-21); 14.2
(C-200); 13.4 (C-20). The same method was applied for the prep-
aration of L3.
appropriate concentration (1 or 10 mM) with culture media,
added to each well and incubated for 3 days. Following each
treatment, 20 L MTS was added to each well and incubated for
m
4 h. MTS is converted to water-soluble blue formazan by a dehy-
drogenase enzyme present in metabolically active cells. Subse-
quently, the plates were read at 490 nm using a microplate
reader (Molecular Devices, Sunnyvale, CA). The results were
reported as the percentage of cell proliferation inhibition
compared to the control (basal cell proliferation ¼ 100%).
L3: 4,40-(4,5-dihydroxy-1,3-phenylenebis(iminomethyl)-bis(1,5-
dimethyl-2-phenyl-1H-pyrazol-3(2H)-one)
Yield: 80%; M.p. 199–200 ꢀC. Anal. Calc. (%) for C30H28N6O4
(536.58 g/mol): C-67.15; N-15.66; Found: C-67.36; N-15.32; IR
(cmꢁ1, KBr): 1610 (m, C]N), 1655 (s, C]O), 1138 (Ar-OH); 1H-NMR
(DMSO-d6, d
, ppm): 2.16 (s; 3H; H-19); 2.42 (s; 3H; H-190); 3.13 (s;
4.3. Antibacterial activity
3H; H-20); 3.21 (s; 3H; H-200); 7.29-7.57 (m; 10H; H-7-11; H-70-
110); 7.60 (s; 1H; H-14); 7.88 (s; 1H; H-18); 9.45 (s; 1H; H-12); 9.70
(s; 1H; H-120).
Qualitative determination of antimicrobial activity was done
using the disk diffusion method. Suspensions in sterile peptone
water from 24-h cultures of microorganisms were adjusted to 0.5
McFarland. Muller–Hinton Petri dishes of 90 mm were inoculated
using these suspensions. Paper disks (6 mm in diameter) contain-
13C-NMR (DMSO-d6; , ppm): 162.5 (C-3; C-30); 160.1 (C-12; C-
d
120); 154.6 (C-16); 150.8 (C-5; C-50); 147.5 (C-15); 133.5 (C-6; C-60);
128.9 (C-8; C-10; C-80; C-100); 127.9 (C-13); 123.7 (C-7; C-11; C-70;
C-110); 122.9 (C-9; C-90); 122.6 (C-18); 120.4 (C-17); 117.9 (C-14);
110.8 (C-4; C-40); 34.2 (C-200); 33.0 (C-20); 13.8 (C-190); 13.1 (C-19).
ing 10
2048
m
L of the substance to be tested (at a concentration of
m
g/mL in DMSO) were placed in a circular pattern in each
inoculated plate. Incubation of the plates was done at 37 ꢀC for 18–
24 h. DMSO impregnated discs were used as negative controls.
Toxicity tests of the solvent, DMSO, showed that the concentrations
used in antibacterial activity assays did not interfere with the
growth of the microorganisms. Reading of the results was done by
measuring the diameters of the inhibition zones generated by the
test substance. Tetracycline and fluconazol were used as reference
substances.
Determination of MIC was done using the serial dilutions in
liquid broth method. The materials used were 96-well plates,
suspensions of microorganism (0.5 McFarland), Muller-Hinton
broth (Merck) and stock solutions of each substance to be tested
4.1.2. General procedure for the preparation of the metal complexes
(1–11)
Synthesis of the copper(II) complexes with 4-(2-hydrox-
ybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one.
An ethanol solution (15 mL) of Schiff base L1 (0.307 g, 1 mmol)
was added to CuSO4$5H2O (0.250 g, 1 mmol) dissolved in
distilled water (10 mL). This solution was refluxed for 2 h and left
at room temperature for 3 days. A brown-red precipitate was
formed for 1. The product was separated by filtration, purified by
washing with cold methanol and then with ether. If a methanol
solution of the Schiff base is used, the obtained product 2 is
green.
(2048
substances to be tested were obtained in the 96-well plates: 1024,
512, 256, 128, 64, 32, 16, 8, 4 and 2
g/mL. After incubation at 37 ꢀC
mg/mL in DMSO). The following concentrations of the
All other complexes were synthesized by the same method. The
elemental analysis confirms the molecular formula. The physical
and analytical data are presented in Table 1.
m
for 18–24 h, the MIC for each tested substance was determined by
microscopic observation of microbial growth. It corresponds to the
well with the lowest concentration of the tested substance where
microbial growth was clearly inhibited.
4.2. Cytotoxicity assay
4.2.1. Ppreparation of test solutions
Stock solutions of the investigated compounds (L1–L4, (1), (6–
12)) were prepared in dimethylsulfoxide (DMSO) at a concentration
of 10 mM and diluted with nutrient medium to various working
concentrations. DMSO was used instead of ethanol due to solubility
problems.
Acknowledgement
The authors thank the Organic of Chemistry Department at
ICECHIM Bucharest, Romania for microanalysis, Horia Hulubei
National Institute of Physics and Nuclear Engineering Bucharest for
help with EPR spectroscopy and Medicines Control Laboratory
Bucharest for antimicrobial activity assays. Thank also to Jenny Roy
who performed the HL-60 cell proliferation assay.
4.2.2. Cell culture
Human promyelocytic leukemia cells HL-60 (ATCC, Rockville,
MD, USA) were routinely grown in suspension in 90% RPMI-1640

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