Biological activity and coordination modes of copper(II) complexes of Schiff base-derived coumarin ligands

Article abstract of DOI:10.1039/c0dt00068j

The coordination modes of copper(ii) complexes of Schiff base-derived coumarin ligands, which had previously shown good anti-Candida activity, were investigated by pH-potentiometric and UV-Vis spectroscopic methods. These studies confirmed the coordination mode of the ligands to be through the N of the imine and deprotonated phenol of the coumarin-derived ligand in solution. In addition, the more active complexes and their corresponding ligands were investigated in the presence of copper(ii) in liquid and frozen solution by ESR spectroscopic methods. A series of secondary amine derivatives of the Schiff base ligands, were isolated with good solubility characteristics but showed little anti-Candida activity. However, cytotoxicity studies of the secondary amines, together with the copper complexes and their corresponding ligands, against human colon cancer and human breast cancer cells identified the chemotherapeutic potential of these new ligands. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0dt00068j

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Biological activity and coordination modes of copper(II) complexes of Schiff
base-derived coumarin ligands†
c
d
c
Bernadette Sarah Creaven,*a,b Eszter Czegle´di, Michael Devereux, Eva Anna Enyedy,
´
Agnieszka Foltyn-Arfa Kia,a,b Dariusz Karcz,a,b Andrew Kellett,d Siobha´n McClean,a,b No´ra Veronika Nagy,e
Andy Noble,g Antal Rockenbauer,e Tere´zia Szabo´-Pla´nka* f and Maureen Walsha,b
Received 4th March 2010, Accepted 13th August 2010
DOI: 10.1039/c0dt00068j
The coordination modes of copper(II) complexes of Schiff base-derived coumarin ligands, which had
previously shown good anti-Candida activity, were investigated by pH-potentiometric and UV-Vis
spectroscopic methods. These studies confirmed the coordination mode of the ligands to be through the
N of the imine and deprotonated phenol of the coumarin-derived ligand in solution. In addition, the
more active complexes and their corresponding ligands were investigated in the presence of copper(II) in
liquid and frozen solution by ESR spectroscopic methods. A series of secondary amine derivatives of
the Schiff base ligands, were isolated with good solubility characteristics but showed little anti-Candida
activity. However, cytotoxicity studies of the secondary amines, together with the copper complexes and
their corresponding ligands, against human colon cancer and human breast cancer cells identified the
chemotherapeutic potential of these new ligands.
particularly as antibiotics such as novobiocin or clorobiocin.1
Introduction
Coumarin derivatives of both natural and synthetic origin have
Coumarin derivatives are well known to possess a diverse array of
also been found to be selectively active against malignant cell
lines including malignant melanomas or renal cell carcinomas.2
Coumarin derivatives are also of interest to our group, and
we have already reported a number of novel coumarin based
compounds with promising antimicrobial activity.3 In particular, a
series of 7-amino-4-methylcoumarin-derived Schiff bases and their
Cu(II) complexes were found to possess significant activity against
Candida albicans with a number of these compounds displaying
anti-Candida activity comparable to that of the commercially
available antifungal drug, Amphotericin B.4 The activities of the
Cu(II) complexes were significantly higher than those of their
corresponding Schiff base ligands.
Whilst the bioactivity of many coumarin derivatives appears to
be enhanced by complexation to a metal ion and this has been
shown by a number of workers over the years,5 the relatively poor
solubility is often a feature of these complexes. In particular a
recent study by our group revealed that the ligands 1a–1k, shown
in Fig. 1, had good activity against C. albicans and this activity
was improved significantly when the ligands were complexed to
pharmacological and biochemical properties and has resulted in
the chemistry of coumarins becoming well established. Currently
some coumarin derivatives are available as prescription drugs,
aDepartment of Science, Institute of Technology Tallaght, Dublin 24, Ireland
bCentre for Applied Science and Health, Institute of Technology Tallaght,
Dublin 24, Ireland
cDepartment of Inorganic and Analytical Chemistry, University of Szeged,
Szeged, Hungary
dThe Inorganic Pharmaceutical and Biomimetic Research Group, Focas
Institute, Dublin 2, Ireland
eChemical Research Center, Institute of Structural Chemistry, Hungarian
Academy of Sciences, Budapest, Hungary
f
Department of Physical Chemistry and Materials Sciences, University of
Szeged, Szeged, Hungary
gDepartment of Chemistry, University of Otago, P.O. Box 56, Dunedin, New
Zealand
† Electronic supplementary information (ESI) available: Experimental
details. CCDC reference number 767589. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c0dt00068j
Fig. 1 Structures of Schiff bases 1a–1k and their corresponding secondary amines 3a–3k showing the numbering system used in the assignment of 1H
and 13C NMR spectra.
10854 | Dalton Trans., 2010, 39, 10854–10865
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Cu(II) ions.4 However, the resulting complexes were difficult to
characterise in solution phase and their antifungal testing proved
problematic for some of the complexes. Nevertheless the significant
anti-Candida potential exhibited by coumarin-derived Schiff bases
1a–1k and their Cu(II) complexes 2a–2k lead us to firstly try
to develop a modified series of complexes with better solubility
characteristics and determine their antifungal as well as their
antibacterial and cytotoxic potential and secondly to undertake
solution thermodynamic studies on a number of active complexes.
pH-potentiometry, frequently combined with UV-Vis spec-
trophotometry is known to be essential for clearing up the com-
positions of the various complexes formed in aqueous solution at
various metal ion, H+ and ligand concentrations, and determining
their formation constants, which result in the speciation under
various experimental conditions. Computer simulation of ESR
spectra in frozen solution reveals important details of coordination
modes.6 The combination of all these methods seems to offer
detailed information on the species formed in solution, which may
play a role in the biological activity of the compounds studied.
Thus in this study we prepared a new series of ligands, 3a–3k, by
reduction of the Schiff base ligands previously studied and isolated
readily soluble secondary amine ligands. We wished to determine
whether the reduction of the imine bond alters the biological effect
significantly or not. These new ligands were assessed for their anti-
Candida activity and all of the ligands and the copper(II) complexes
were assessed for their antimicrobial and anticancer potential. In
addition we aimed to recognize the nature of the complexes in
solution, considering the better biological relevance of the liquid
phase compared to the crystalline phase. Solution equilibria of
the Schiff-base ligands 1a–1f, both in the absence and presence
of Cu(II) ions were studied by the pH-potentiometric and UV-Vis
spectroscopic methods, and some of the active complexes and the
corresponding ligands in the presence of Cu(II) were investigated
in liquid and frozen solution by ESR and are reported herein.
Table 1 Starting Schiff base and identification of functional groups on
Schiff bases 1a–1k and secondary amine derivatives 3a–k
Starting Schiff base Secondary amine R1
R2
R3
1a
1b
1c
1d
1e
1f
1g
1h
1i
3a
3b
—
3d
3e
—
3g
3h
3i
–H
–OH
–H
–H
–H
–OH
–H
–H
–H
–H
–H
–H
–H
–OCH3
—
–OCH2CH3 –OCH3 –H
–H
–H
–H
–H
–H
–H
–Cl
–Br
–I
–NO2
–NO2
–Cl
–Br
–I
–H
–OCH3
1j
1k
3j
3k
80% (w/w) d6-DMSO/D2O mixture at a concentration of ~2 mM
without further modification of the pH. Microanalytical data were
provided by the Microanalytical Laboratory, National University
of Ireland, Belfield, Dublin 4.
Synthesis of Schiff base ligands 1a–1k and their Cu(II) complexes
2a–2k
The synthesis of all of the ligands 1a–1k and their Cu(II) complexes
were previously reported.4
Synthesis of Schiff base-derived secondary amines 3a–3k
All of the Schiff base derived secondary amines 3a–3k were
synthesised using the following general procedure. NaBH4 (1.1
mmol) was slowly added to a warm solution (40 ◦C) of an
appropriate Schiff base (1.1 mmol) in ethanol (~50 ml). The
solution was then heated to reflux for 30 min, allowed to cool
to ambient temperature, acidified to pH = 4.5 (litmus paper) using
trifluoroacetic acid and then refluxed for a further 30 min. Excess
solvent (~ 35 ml) was then removed under reduced pressure. A light
precipitate was collected by filtration, washed with cold methanol
(2 ml) and allowed to air dry. Assignments of NMR spectra of
the ligands are based on the numbering scheme shown in Fig. 1.
Substituents R1, R2, and R3 of the aldehyde moiety are given in
Experimental
Materials/instrumentation
1
Table 1. All of the ligands were fully characterised by H, 13C
All chemicals were purchased from Sigma-Aldrich and all except
salicylaldehyde and aniline were reagent grade and used without
purification. The salicylaldehyde and aniline were of puriss quality.
Their purities were checked and the exact concentrations of the
prepared stock solutions were determined by the Gran method.7
A CuCl2 stock solution was made using anhydrous CuCl2 and its
concentration was determined by complexometry via the EDTA
complex. Infrared spectra were recorded in the region of 4000 cm-1
to 400 cm-1, on a Nicolet Impact 410 Fourier-Transform Infrared
(FITR) spectrophotometer using Omnic software. All infrared
spectra were run as KBr discs. Melting point values were recorded
on a Stuart scientific SMP1 melting point apparatus. Values were
NMR, IR, and UV-Vis spectroscopies as well as by melting point
and elemental analysis (Tables 2 and Tables S1–S3†).
X-ray crystallography
X-ray crystallographic studies were performed at the University of
Otago, New Zealand on a Bruker Kappa Apex II diffractometer
with a CCD area detector. The crystal structure was solved using
SHELXS-97. All structures were refined against F2 using all
data by full-matrix least-squares techniques with ShelX-86 and
SHELXL-97.8 Crystal data and structure refinement of 1e are
given in Table S4.†
◦
1
taken up to 320 C. All H NMR spectra were recorded in the
region of -5 ppm to 15 ppm from TMS with a resolution of 0.18 Hz
or 0.0006 ppm. All 13C-NMR spectra were recorded in the region
-33 ppm to 233 ppm from TMS with a resolution of 0.008 ppm.
Spectra were run on a JEOL JNM-LA300 FT-NMR (300 MHz
1H and 75 MHz 13C) in d6-dimethyl sulfoxide(DMSO). For the
potentiometric-related 1H NMR studies, a Bruker Ultrashield 500
Plus instrument was used and the ligands were dissolved in an
Anti-Candida susceptibility testing
The ligands 3a, 3d, 3e, 3g–3i, 3k and a commercially available
drug Amphothericin-B were tested against a clinical isolate of
the fungal strain, Candida albicans (ATCC 10231). The anti-
Candida activities were determined using a broth microdilution
susceptibility protocol (NCCLS).9Thescreeningprotocol included
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Table 2 Physical data for the secondary amines 3a–3k
Found (Calc) (%)
Amine
Mw/g mol-1
Yield (%)
C
H
N
Empirical formula
m.p./◦C
3a
3b
3ca
3d
3e
3f*
3g
3h
3i
281.31
297.31
—
311.33
325.36
—
350.20
439.10
533.10
326.30
356.33
73
72
—
73
79
—
89
79
86
82
54
72.32 (72.58)
64.20 (68.68)
—
69.19 (69.44)
70.00 (70.14)
—
58.09 (58.31)
46.63 (46.50)
38.24 (38.30)
59.80 (62.57)
59.81 (60.67)
5.35 (5.37)
4.92 (5.09)
—
5.50 (5.50)
5.79 (5.89)
—
3.67 (3.74)
2.92 (2.98)
2.42 (2.46)
4.16 (4.32)
4.40 (4.53)
5.13 (4.98)
4.56 (4.71)
—
4.60 (4.50)
4.47 (4.31)
—
4.09 (4.00)
3.23 (3.19)
2.87 (2.63)
8.17 (8.59)
7.71 (7.86)
C17H15NO3
C17H15NO4
—
C18H17NO4
C19H19NO4
—
C17H13NO3Cl2
C17H13NO3Br2
C17H13NO3I2
C17H14N2O5
C18H16N2O6
215 – 217
181 – 184
—
212 – 213
182 – 184
—
220 – 222
218 – 220
203 – 205
217 – 221
201 – 203
3j
3k
a Not isolated.
the following steps: Isolates were grown for 24 h on Sabouraud
dextrose agar (SDA) plates at 37 ◦C. Cell suspensions were
prepared in sterile phosphate buffered saline (PBS) to a density
equal to a 1.0 McFarland standard yielding a concentration of
1 ¥ 106 cells ml-1 and then further diluted (1 : 100) with minimal
media (MM). The ligands and the commercial drug were prepared
as 1% solutions in DMSO. All of the compounds were tested at a
range of concentrations of 20, 10, 5 and 1 mg ml-1, and the plates
were incubated for 24 h at 37◦ C. Each compound was assessed in
triplicate and three independent experiments were performed.
using methylthiazolyldiphenyl-tetrazolium bromide-based assay
(MTT).10 Cells were seeded at 4 ¥ 105 cells ml-1 in 96-well plates
and incubated at 37 ◦C in 5% CO2 for 24 h. Cells were treated
with a four-log range of concentrations of the test compounds
in triplicate from 0.1 to 100 mM or with a solvent control (0.5%
DMSO) in complete medium. Following 24 h incubation, cells
were incubated with 20 ml of MTT (5 mg ml-1) in 0.1 M PBS,
pH 7.4 at 37 ◦C in a humid atmosphere with 5% CO2 for 4 h.
Media was then gently aspirated from the test cultures and 100 ml
of DMSO was added to all wells to solubilize the MTT crystals.
The plates were then shaken for 2 min and the absorbance was read
at 550 nm in a microtiter plate reader. IC50 values were determined
and defined as the concentrations of test compound required to
reduce the absorbance of the MTT-formazan crystals by 50%,
indicating 50% reduction in cellular metabolic activity.
Antibacterial susceptibility testing
The ligands and complexes were tested against two species of
bacteria, namely Escherichia coli (NCIMB 9485) and Staphylo-
coccus aureus (NCIMB 12702). E. Coli was cultured in Luria-
Bertani (LB) broth purchased from Sigma-Aldrich. S. aureus was
cultured in Tryptone Soy Broth (TSB) purchased from LabM,
UK. Both strains were provided by the National Collection of
Industrial, Marine and Food Bacteria. The commercial antibiotics
gentamicin and amikacin were used as positive controls. Minimum
inhibitory concentrations (MIC) were determined by the broth
microdilution method.9 Stock solutions of each compound to be
tested were prepared in DMSO and serially diluted in triplicate
using broth (100 ml/well) at a concentration range of 6.25 to 200
mM. The DMSO was diluted to 0.5% for all concentrations of
compound tested. Bacteria were also treated with a solvent control
(0.5% DMSO). Overnight cultures (10 ml) of bacterial strains were
inoculated in broth (100 ml) and incubated at 37 ◦C with agitation
until the optical density at 600 nm (O.D.600) reached approximately
0.6. The cultures were then diluted to 1 ¥ 106 CFU/ml and seeded
at 100 ml/well in 96 well plates in the presence of a range of
concentrations of test compound in triplicate. Following a 24 h
incubation the absorbance was read at 600 nm in a microtiter
plate reader. The MIC90 was defined as the concentration of test
compound which inhibited 90% visible growth of the species in
24 h.
1H NMR measurements with Schiff-base ligands in solution
1H NMR studies were carried out on a Bruker Ultrashield 500 Plus
instrument at ~2 mM ligand concentration, the pH was not altered
after dissolution. The aim of these measurements was to check
the hydrolytic stability of the ligands 1a–1f. No spectral changes
were observed in the first three hours. Then the characteristic
peak of the aldehyde H appeared at 10.09 ppm as a result of
the decomposition. Therefore, the stock solutions of the ligands
were always made freshly and used within three hours to avoid
hydrolytic decomposition.
pH-Potentiometric studies
The pH-potentiometric measurements were carried out at 25.0
0.1 ◦C, at an ionic strength of 0.1 M (KCl) in the pH range 2.5–16.0
(corresponding to the high pKw of the solvent mixture, see below).
The ligand concentration varied in the range 0.8–2 mM, and the
Cu(II) to ligand ratios varied between 1 : 1–1 : 4. The titrations
were performed with a carbonate-free KOH solution (0.1 M).
Both the base and the HCl used were Sigma-Aldrich products
and their concentrations were determined by pH-potentiometric
titrations. The initial volume of the samples was 5.0 ml. Samples
were completely deoxygenated by bubbling purified argon through
them for ~20 min before the measurements, and argon was also
passed above the solutions during the titrations. An Orion 710A
pH meter equipped with a Metrohm combined electrode (type
Cytotoxicity studies
All ligands and complexes were screened for their cyto-
toxic activity against two cell lines, namely HT29 (human
colon cancer cells) and MCF-7 (human breast cancer cells)
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6.0234.100), and a Metrohm 665 Dosimat burette were used.
The electrode system was calibrated according to the Irving
method.11The average water ionization constant, pKw = 18.40
0.05, obtained in 80% (w/w) DMSO/water12 was used in the
calculations carried out with the programs SUPERQUAD13 and
PSEQUAD.14 The accepted fitting of the titration curves was
always less than 0.01 ml.
The anisotropic ESR spectra were evaluated by using the EPR
program which allows for the description of the experimental
spectra as superposition of two component curves.15 Either axial
or rhombic g-, hyperfine, and quadrupole coupling tensors were
assumed. The magnetic quantum number-dependent linewidths
WMI of the copper hyperfine multiplet were calculated from
the relaxation parameters a, b and g as WMI = a + bMI
+
2
The overall (concentration) stability constant b(MpLqHr) is
defined for the general equilibrium pM + qL + rH ꢀ MpLqHr as
b(MpLqHr) = [MpLqHr]/[M]p[L]q[H]r where M denotes the metal
ion and L the completely deprotonated ligand. The neutral form
of ligands 1a, 1b and 1d–1f is [HL], while it is symbolized by [H2L]
for 1c.
g MI (MI is the magnetic quantum number of copper nuclei).
Anisotropy of the relaxation parameters a, b and g also was
taken into consideration. As we used a natural mixture of copper
isotopes, the spectra were calculated as the sum of the curves
of molecules containing isotope 63Cu or 65Cu weighted by their
natural abundances. The hyperfine coupling constants refer to the
isotope 63Cu, and are given in gauss (G) units throughout the paper
(1 G = 10-4 T).
The quality of fit was characterized by the noise-corrected
regression parameter R computed from the average square de-
viation for the curve in question. The noise was deduced from the
quadratic error of the fit to obtain R = 1 for a perfect fit.
Spectrophotometric and spectrofluorimetric measurements
The UV-Vis spectrophotometric measurements were performed
at 50 mM ligand concentration in the pH range 2–16. For the
samples containing Cu(II), 1 mM Cu(II) concentration at metal-to-
ligand ratios of 1 : 1 and 1 : 2 were applied. A Unicam Helios Alpha
spectrophotometer was used to record the spectra in the range
250–800 nm. The path length was 1 cm. The proton dissociation
constants were calculated by the computer program PSEQUAD.14
The pH-dependent fluorescence measurements were carried
out on a Hitachi-4500 spectrofluorimeter with an excitation
wavelength set at 355 nm, and the emission spectra were recorded
in the range 380–550 nm using 10 nm/10 nm slit width in 1 cm
quartz cell at 25.0 0.1 ◦C. Samples contained 0.2 mM Schiff base
ligand at 0.1 M (KCl) ionic strength. Three-dimensional spectra
were recorded at 200–450 nm excitations, and at 250–550 nm
emission wavelengths.
Results and discussion
The synthesis and structural characterisation of a series of Schiff
base ligands 1a–1k and their corresponding Cu(II) complexes 2a–
2k was previously reported.4 As Schiff bases 1a and 1e were
obtained as crystalline products suitable for X-ray analysis, their
crystal structures were determined and reported in the ESI (Table
S4 and Fig. S1†).
Synthesis and characterisation of Schiff base derived secondary
amines
The secondary amine products were obtained by refluxing
ethanolic solutions of the Schiff bases in the presence of an
equimolar amount of sodium borohydride (NaBH4). Reduction of
compounds 1a–k yielded a series of nine secondary amines namely
3a, b, d, e, and 3g–k. Elemental analysis indicated that the majority
of the secondary amines were isolated with acceptable purity,
though repeated attempts to isolate pure samples of compounds
3b and 3j were unsuccessful and those two compounds were not
sent forward for biological screening. Isolation of the hydroxy and
methoxy substituted compounds, 3c and 3f was not successful.
Physical data and empirical formulae are presented in Table 2. All
of the secondary amine products were readily soluble in a range
of organic solvents. IR, 1H and 13C NMR spectral data are given
in the ESI, Tables S1–S3.†
ESR measurements at 298 K
The spectra were recorded in a circulating system, in the course
of titrations which were carried out in an air-conditioned room at
298.0 0.2 K. The solutions were kept under an argon atmosphere,
the pH was varied in the range 2–15 at a total (analytical) copper(II)
concentration TCu = 1 mM and a total ligand concentration TL =
1 mM, and TCu = 0.5 mM and TL = 1 mM. We applied 80% (w/w)
DMSO/water as solvent, 0.2 M KCl as background electrolyte, the
pH was adjusted with HCl (0.2 M) and then NaOH (0.5 M) to an
accuracy of 0.01 pH unit, using a Metrohm 765 Dosimat apparatus
equipped with a Metrohm LL combined micro electrode. A
Masterflex CL peristaltic pump ensured the circulation of the
solution through the capillary tube in the cavity. The ESR spectra
were recorded after 2 min of circulation at the chosen pH values,
using an X-band Bruker EleXsys E500 instrument.
The IR spectra of the Schiff bases consisted of broad bands
in the region of 3400–3100 cm-1 assigned to the hydroxy groups,
which were intramolecular H-bonded to the imine nitrogen.4 In
the spectra of the secondary amines, this region was occupied by a
number of bands of varying intensity, corresponding to the N–H
symmetrical and asymmetrical stretching vibrations of secondary
amines, as well as aromatic C–H stretching vibrations. Another
medium intensity band in the range of 1529–1444 cm-1 was
attributed to N–H bending (scissoring) vibrations of the amine.
Significant changes were also observed in the positioning of the
lactone C O stretching vibration and the phenolic C–O stretch.
The spectra of the copper(II) complexes dissolved in 80% (w/w)
DMSO/water mixture were also recorded. The concentration of
the complexes was 0.5 mM. The pH of the solutions was measured
as above.
ESR measurements and analysis of spectra at 77 K
Samples of 0.15 ml volume, taken at various pH from the mixtures
titrated at 298 K, or from the solutions of the complexes, were
frozen in liquid nitrogen. Then the ESR spectra were recorded at
77 K with the same spectrometer as above.
1
The H- and 13C NMR spectra supported the formation of
the secondary amine group in the synthesised derivatives by the
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Table 3 Activity of Schiff bases 1a–1k, Copper(II) complexes 2a–2k4 and secondary amines 3a,3d,3e, 3g–3i, 3k against C. albicans expressed as MIC50
values [mM]
Schiff base
MIC50/mM
Complex
MIC50/mM
Secondary amine
MIC50/mM
>100
1a
92.3
35.4
89.2
62.0
92.6
44.5
17.1
3.2
1.2
9.1
95.4
0.7
4.7
2a
2b
2c
2d
2ea
2f
2g
2h
2i
5.2
3a
1b
10.4
16.7
8.2
—
14.5
3.6
4.4
0.7
9.8
12.6
1c
1d
3d
3e
>100
>100
1e
1f
1g
3g
3h
3i
>100
>100
>100
1h
1i
1j
2j
2k
1k
3k
>100
Amphotericin B
Ketokonazoleb
a Not tested. b Value recorded previously in our lab under the same conditions.7
disappearance of the imine (–CH N–) singlet and the appearance
of two new signals attributed to the –CH2–NH– moiety. A doublet
at approximately 4.35 ppm and a relatively broad triplet in the
region of 7.0–7.3 ppm corresponded to the methylene and amine
groups, respectively. As expected, the only major change that was
observed in the 13C NMR spectra of the secondary amines, relative
to their corresponding Schiff base, was an upfield migration of the
C13 peak.
The poor solubility of both the ligands 1a–1k and the complexes
2a–2k ledus tothesecondaryamines as more solubleligands which
we hoped would lead to the isolation of a new series of Cu(II)
complexes. Although the reduction of the imine bond resulted
in a series of readily soluble secondary amines, unfortunately the
isolation of their subsequent complexes was unsuccessful. In most
cases the formation of expected products did not occur or the
isolated products were of low purity and proved difficult to isolate
in pure form.
Antibacterial activities of Schiff base ligands 1a–1k, Cu(II)
complexes 2a–2k, and secondary amines 3a, 3d, 3e, 3g–3i, 3k
The anti-Candida activity of the secondary amines, their corre-
sponding Schiff bases, and copper(II) complexes has shown the
therapeutic potential of these compounds. Many of the complexes
we have previously investigated have shown a broad range of
activity3,16 and thus the therapeutic potential of all compounds
was further investigated. The antibacterial activities of 1a–1k, 2a–
2k and 3a, 3d, 3e, 3g–3i, 3k as well as a copper(II) salt in DMSO
were assessed as described in the Experimental section and all were
found to be inactive. Inactivity against both Gram-negative and
Gram-positive species recorded for 1a–k, 2a–k together with their
significant anti-Candida activity suggested their high selectivity
against fungal pathogens. The selectivity might be as a result of
the well-known structural differences between fungal and bacterial
cells,17 although the exact reasons remain unclear as yet.
Anti-Candida activities of Schiff base ligands 1a–1k, Cu(II)
Cytotoxicity studies
complexes 2a–2k, and secondary amines 3a, 3b, 3d, 3e, 3g–3k
A number of coumarin derivatives, including Cu(II) complexes,
previously isolated by our group have shown considerable anti-
cancer activity against a number of cell lines.17 All of the ligands,
a copper(II) salt and the copper complexes were assessed for their
cytotoxicity against two human derived cell lines namely HT29
(human colon cancer cells) and MCF-7 (human breast cancer
cells) using methylthiazolyldiphenyl-tetrazolium bromide-based
assay (MTT). The IC50 values (determined as the concentration
of compound required to reduce cellular viability by 50%) for the
ligands and their complexes for the colon cancer cell line showed
that the copper(II) salt, ligands 1a–1k and their Cu(II) complexes
were not cytotoxic to these cells. The positive control used in
this study was mitoxantrone, a drug HT29 cells are known to
be sensitive to.18 Interestingly cisplatin, which is often used as a
positive control for this type of study, also had an IC50 value greater
than 100 mM. However, whilst most of the secondary amines
did not display any anticancer activity the halogen-substituted
secondary amines, 3g, 3h and 3i did show cytotoxicity to the HT29
cell line (IC50 values of 81.5 ( 7) mM, 60.3 ( 3) mM and 65.8 ( 10)
mM respectively, which were comparable to that of mitoxantrone
(76.9 ( 6) mM).
The previously reported anti-Candida activities of 1a–1k and their
corresponding Cu(II) complexes 2a–2k prompted the investigation
into the anti-Candida activity of the secondary amine ligands.4 The
anti-Candida activities of Schiff bases 1a–1k, Cu(II) complexes 2a–
2k, and secondary amines 3a, 3d, 3e, 3g–3i and 3k (expressed as
MIC50 values, the minimum concentration required to inhibit 50%
of cells growth) are given in Table 3. The anti-Candida activities of
the secondary amines were significantly lower than the compounds
tested in the previous study, whose activity was comparable to
that of the commercially available antifungal drugs, Amphotericin
B and Ketoconazole. Solubility issues were not considered as a
contributing factor to the decreased activities of the secondary
amines, as all of the tested compounds were highly soluble in
DMSO, and the addition of the Candida albicans suspension did
not cause their precipitation. The relatively low activities of all the
secondary amines in comparison to the Schiff bases, might suggest,
that the presence of the imine moiety is crucial for high anti-
Candida activity and that the Schiff bases are more active because
they can form complexes more easily than their corresponding
secondary amines.
10858 | Dalton Trans., 2010, 39, 10854–10865
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Against the human breast cancer cell line MCF-7 the range of
cytotoxic activity recorded for Cu(II) complexes 2a–2k was similar
to that of Schiff bases 1a–1k, but a few compounds in this series
exhibited slightly higher toxicity than their corresponding free
ligands. The most significant results were recorded for compounds
2i and 2k when tested against the MCF-7 cells. These compounds
showed IC50 values of 79.8 ( 5) mM and 34.5 ( 6) mM respectively,
while IC50 values for their corresponding free ligands 1i and
1k were above 100 mM. MCF-7 cells remained unaffected by
the secondary amines except for compound 3g, the dichloro-
substituted derivative, which exhibited an IC50 value of 84.0 ( 2)
mM as compared to an IC50 value of 44 ( 3) mM for mitoxantrone,
which is approved for use in the treatment of metastatic breast
cancer.
Scheme 1 Deprotonation steps for (a) 1c and (b) 1a, 1b and 1d–1f.
Thus, while the presence of the imine moiety seems to be
important for high anti-Candida activity this study has indicated
the possible usefulness of the secondary amines in the design of
novel anticancer agents. The halogen-substituted secondary amine
derivatives had activity equal to or better than the commercial
drug used to treat human colon cancer. In addition the solubility
profile of the secondary amines is much better than their corre-
sponding imines. For the Schiff bases studied here the presence of a
copper ion did not increase the cytotoxic activity of the compound.
The anti-Candida, antimicrobial and cytotoxicity studies had
shown that the ligands and copper(II) complexes studied here did
have therapeutic potential and we were interested in studying the
likely behaviour of these complexes and ligands at physiological
pH. Thus, aqueous solution studies were performed on six 4-
methyl-coumarin Schiff-base derivatives (1a–1f) related to (i)
their solubility in solvent mixtures of DMSO and water, (ii)
their stability in 80% (w/w) DMSO/H2O solvent mixture by
1H NMR studies, (iii) their proton dissociation processes by pH-
potentiometry, UV-Vis spectrophotometry and spectrofluorimetry
(iv) their complex formation with Cu(II) by pH-potentiometry,
UV-Vis spectrophotometry and ESR spectroscopy in 80% (w/w)
DMSO/H2O.
A representative three-dimensional spectrofluorimetric spec-
trum recorded for 1c is shown in Fig. 2a. An intense emission band
can be observed at an excitation maximum ~350 nm. The intensity
of this band exhibits pH-dependence at pH < 4 as can be seen in
Fig. 2b. The changes can be assigned to the proton loss/gain of
the Schiff base N. The proton dissociation constant for the Schiff
base NH+ can be estimated from these spectral changes: its pKa
is ~1.5. As the Schiff base N is in the non-protonated state in the
precisely measurable pH range, in the following sections we took
into consideration merely the deprotonation of the OH groups.
Fig. 2 (a) 3D spectrofluorimetric spectrum of 1c at pH = 1.5, at 0.2 mM
concentration in 80% (w/w) DMSO/H2O; I = 0.1 M (KCl); t = 25.0 ◦C)
max
max
(lEX = 355 nm; lEM = 445 nm); (b) pH dependence of the intensity
of the emission at 455 nm for 1c at 0.2 mM concentration in 80% (w/w)
DMSO/H2O; I = 0.1 M (KCl); t = 25.0 ◦C; lEX = 355 nm.
Proton dissociation processes
First of all the solubility of the ligands were studied, as they are
poorly soluble in water; therefore, various DMSO/H2O solvent
mixtures were tested as possible media for the equilibrium studies.
80% (w/w) DMSO/H2O was found to be suitable to reach the
minimum concentration (1–2 mM) for the ligands 1a–1f, which
is necessary for the pH-potentiometric titrations and the ESR
measurements. This solvent mixture was also applied for the
equilibrium study of other Schiff-bases.19
In principle, the ligands 1a and 1d–1f possess two dissociable
protons, one on the Schiff base N in strongly acidic solution,
the other one is that of the phenolic OH. The ligands 1b and
1c also have a second phenolic OH group in ortho and meta
positions to the first one, respectively (Fig. 1). The Schiff base
[H3L]+ (Scheme 1) is highly acidic, therefore, its proton loss cannot
be measured by pH-potentiometry due to the acidic error of the
glass electrode. Fortunately, its deprotonation can be followed by
spectrofluorimetry, as it is situated near the fluorophor 4-methyl-
coumarin moiety. Using the appropriate excitation, the intensity
of the emitted light was found to be sensitive to the protonation
states of these ligands.
The proton dissociation constants for the phenolic OH groups
of the ligands were determined by both pH-potentiometry
and UV-Vis spectrophotometry. The pH-potentiometric titration
curves reveal one deprotonation step corresponding to the process
characterized by pK1 (Scheme 1). The only exception is 1c, where
a well-separated second step also appears at high pH, which can
be assigned to the proton loss of the second OH (pK2, Scheme 1).
The ligands display intense absorption bands in the UV region
due to the p→p* transitions of the phenyl rings and the extended
conjugated electronic system of the imine and coumarin moieties
(Fig. 3). Further bands appear at l > 300 nm which correspond
to n→p* transitions assigned to the azomethine group.4 Deproto-
nation of the phenolic OH groups results in characteristic spectral
changes for all ligands. Development of a wide band in the 390–
450 nm range (appearance of yellow colour) and a slight red shift
from 350 to 358 nm could be observed at 8.5 < pH < 11. It is
noteworthy that the spectra did not change with time, suggesting
the stability of ligands in the solvent used. All ligands exhibited
similar spectral behaviour, except for 1c. For the latter, the
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methoxy (1d, 1f), and particularly the hydroxyl group (1b and 1c)
results in lower pK1 values (Table 4). The deprotonation of the
second OH group takes place in the well-measurable pH range for
1c, allowing pK2 to be determined. On the contrary, for 1b the
deprotonation of the first phenolic OH allows a strong hydrogen
bond to form between the phenolate O- and the adjacent OH
group, which shifts the proton loss of the latter to higher pH, into
the non-measurable region.
Complex formation with copper(II)
Representative titration curves for the Cu(II)–1e and Cu(II)–
salicylaldehyde systems are shown in Fig. S2.† Almost all the
Schiff bases were able to keep Cu(II) in solution even at low
(1 : 1) ligand-to-metal concentration ratio, while in the case of
salicylaldehyde, precipitation (most probably Cu(II)-hydroxide)
occurred in neutral or slightly basic solutions both at equal ligand
and metal concentrations and in the presence of a 2-fold ligand
excess. In the solution of 1b and Cu(II), the irreversible Cu(II)-
catalyzed oxidation of the pyrocatechol moiety took place at
pH > 12, which was indicated by the red colour of the solution.20
The titration curves suggest the formation of a large amount of
mixed hydroxido complexes in basic solution with all ligands. The
Fig. 3 UV-Vis spectra recorded at different pH values for (a) 1a and (b)
1c at 5.0 ¥ 10-5 M concentration in 80% (w/w) DMSO/H2O; I = 0.1 M
(KCl); t = 25.0 ◦C.
stability constant for the hydrolytic species (log b[Cu(OH)]+
=
-7.8(1)) was also determined and included in the calculation
models, though, practically, it did not affect the fitting parameters
and stability constants, indicating that the formation of this species
was negligible in the presence of the Schiff-base ligands.
The data show the predominant formation of mono-ligand
complexes [CuLH]+, [CuL] and [CuLH-1]- for 1c, while the
predominant species for the other ligands were [CuL]+, [CuLH-1]
and [CuLH-2]-. When the bis-ligand complex [CuL2] was also taken
into consideration, only a slight decrease of fitting parameter
occurred; and, the stability constant of this species could be
determined though with high uncertainty, therefore, this complex
was finally omitted.
[H2L] → [HL]- deprotonation step was accompanied by an
increase in absorption in the range 310–380 nm, while a red shift
and decrease in absorption could be detected at pH > 11 in the
same wavelength range, as the subsequent proton dissociation
([HL]- → [L]2-) took place (Fig. 3b).
The pK1 and pK2 constants are shown in Table 4. The values
obtained by the two different methods are in good agreement. The
substituents affect the proton dissociation constants significantly.
The pK1 value of the non-substituted ligand 1a was found to be
similar to that of salicylaldehyde. The presence of the ethoxy (1e),
Table 4 Proton dissociation constants (pK) of 1a–1f and salicylaldehyde, and stability constants (log b) of their Cu(II) complexes, together with some
stepwise and derived stability constantsa in 80% (w/w) DMSO/H2O at t = 25.0 ◦C; I = 0.10 M (KCl)
1a
1b
1c
1d
1e
1f
Salicyl-aldehyde
pK1
pK2
pK1
pK2
9.66(6)
—
9.65(1)
—
8.79(4)
—
8.60(1)
—
8.79(1)
13.51(2)
8.85(1)
13.52(1)
9.22(5)
—
9.23(1)
—
9.38(2)
—
9.33(6)
—
9.39(2)
—
9.40(9
—
9.65(1)
—
—
b
b
—
log b([CuLH])c
—
7.7(1)
—
—
0.1(1)
—
—
—
—
—
—
—
21.6(1)
—
—
6.9(1)
—
19.71(8)d
—
11.91(8)
—
—
2.47(9)
—
—
—
6.28(9)
—
—
-1.62(9)
—
—
-12.7(1)
—
—
6.30(6)
—
—
-1.71(6)
—
—
—
8.29(9)
—
—
1.4(1)
—
—
-9.9(1)
—
—
6.27(6)
—
—
-1.30(4)
—
—
log b([CuL])c
log b([CuLH(OH)]c
log b([Cu2L2])c
log b([CuL(OH)])c
log b([CuLH(OH)2])c
log b([Cu2L2H-2])c
log b([CuL(OH)2])c
Log b([CuLH-2])c
log b([Cu2L2(OH)4])c
fitting parameter/cm3
-12.73(8)
—
-11.80(8)
—
-7.2(1)
—
-16.9(1)
—
—
—
—
—
—
5.98 ¥ 10-3
4.35 ¥ 10-3
6.53 ¥ 10-3
5.94 ¥ 10-3
8.88 ¥ 10-3
6.54 ¥ 10-3
6.70 ¥ 10-3
log K*e
-1.96
—
-2.59
-2.94
-3.08
-1.10
-3.38
a Standard deviation (SD) of the equilibrium constants is given in parentheses. b Determined by UV-Vis spectrophotometry. c Charges are omitted for
simplicity. d log b([CuLH]) - pK2 = 6.20. e log K* = logb(CuL) - pK1 for all ligands except to 1c where log K* = logb([CuLH(OH)]) - pK1 - pK2.
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For the justification of the equilibrium model, we studied ESR
spectra in the fluid and frozen solutions at various metal and ligand
concentrations and pH for three systems, where the solubilities of
the ligands and the complexes allowed. The spectra are shown in
Fig. S3–S5.† The most striking feature of the spectral series is that
the curves taken at equal metal and ligand concentrations or at
a two-fold excess of ligand at similar pH, are closely similar. If
the bis complex was formed in significant concentration, it would
be accompanied by a new spectral component at ligand excess,
particularly near pH 9. The lack of such a change supports the
pH-potentiometric equilibrium model built exclusively from mono
complexes.
Based on the pH-metric results, it can be concluded that in
the complexes [CuL]+ of 1a and 1d–1f, the phenolic OH group
is most probably deprotonated and bound to Cu(II), together
with the Schiff-base N. As there is no other dissociable proton in
these ligands, [CuLH-1] and [CuLH-2]- are unambiguously formed
by the proton loss of the coordinated water molecules: they are
the mixed hydroxido complexes [CuL(OH)] and [CuL(OH)2]-. In
the case of ligand 1c, the second phenolic OH group is not in a
chelating position, and its possible monodentate coordination is
unlikely to replace the chelate ring. Therefore, this OH probably
releases its proton at high pH as in the absence of Cu(II). Thus
[CuLH]+ of 1c corresponds to [CuL]+ of 1d–1f, and a similar
coordination mode is assumed, moreover [CuL] and [CuLH-1]-
are most probably mixed hydroxido complexes and the more
correct formulae of them are [Cu(LH)(OH)] and [CuLH(OH)2]-,
respectively. For 1b with three donor groups in adjacent positions
on the aromatic ring, a variety of coordination modes may occur,
which is discussed in the next Section.
Some complexes of 1a and 1b proved to be ESR-inactive,
suggesting a strong interaction between the metal ions. On
the basis of this spectroscopic evidence, dimeric species were
included in the speciation models instead of monomeric ones
(Table 4), though the fitting parameters did not show significant
differences: pH-potentiometry cannot distinguish species with the
same stoichiometry (e.g. [CuL(OH)] or [Cu2L2(OH)2]).
The stoichiometries of the Cu(II) complexes of 1a–1f and
salicylaldehyde, and their stability constants giving the best
fit to the pH-potentiometric experimental data, together with
some stepwise and derived equilibrium constants are collected in
Table 4.
Fig.
4
Concentration distribution curves of Cu(II)–ligand 1a (a),
Cu(II)–ligand 1b (b) and Cu(II)–ligand 1c (c) systems (clig = 2.0 mM; cCu(II)
1.0 mM; 80% (w/w) DMSO/H2O; I.
=
The d-d absorption bands in the visible region (not shown)
reflect a stronger ligand field for [CuL]+ of Schiff bases (lmax
650 nm) than the corresponding complex of salicylaldehyde (lmax
~ 690 nm).
~
ESR spectra of the equilibrium systems
The concentration distribution curves calculated from the data
in Table 4 for the Cu(II)–1a–1c systems are illustrated in Fig. 4.
The complex formation for 1b starts at significantly lower pH
than for the other ligands, indicating the overriding stability of its
complexes.
The derived stability constant, log K* refers to the Cu(II) + [HL]
ꢀ [CuL] + H competitive equilibrium. For 1a, and particularly
1f, the higher log K* (Table 4) represents a more favoured chelate
formation in [CuL]+ compared to the proton complex, while
for the other ligands log K* is lower and does not show large
differences.
The value of log K* is somewhat higher for 1a–1f than for
salicyaldehyde, most probably due to the higher stability of the
(O-,N) chelate ring. For 1d and 1e, where a methoxy or an ethoxy
group is bound in the R1 position, log K* values are the lowest,
most likely as a result of the repulsive steric effect of the adjacent
groups.
Our aim was to gain information on typical coordination modes
in solution. The non-substituted Schiff base 1a and the OH-
substituted 1c, where the OH groups are in meta positions to
each other, are typical examples regarding the stabilities of their
complexes, while 1b containing two neighbouring phenolic OH
groups forms fairly more stable complexes than the other ligands
(Fig. 4, Table 4). Unfortunately, the spectra in liquid solution
are broad, poorly resolved and irregularly shaped (Fig. S3–
S5†). We explain this fact by the slow rotation of molecules
in the solvent mixture of high viscosity, which is insufficient
to average the anisotropy of magnetic parameters completely,
causing intermediate signal shapes. Therefore, the isotropic ESR
parameters cannot be determined in fluid solution. The spectra in
frozen solution, however, are well-resolved (Fig. S3–S5†). In the
case of those solutions, where only one or two species are formed
in considerable concentrations, they can be analysed uniquely, the
fit between the experimental and calculated curves is good (Fig. 5).
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Fig. 5 Selected ESR spectra for the Cu(II)–1c equilibrium system in frozen
solution at 77 K, TCu = 0.5 mM, TL = 1 mM. Black: experimental curves,
red: calculated spectra.
For the selection of suitable spectra, the concentration distribu-
tion curves obtained by pH-potentiometry in liquid solution could
be used, indicating that freezing of the solution does not induce
significant changes in the speciation. The ESR parameters for the
complexes formed in solution are shown in Table 5.
Coordination modes in solution
For 1a and 1c, the only (chelating) binding site is a “Schiff-base-
like” one: they can coordinate to Cu(II) through the N and the
phenolate O- atoms. This mode of ligation is characteristic of
[CuL2] in the crystalline state4 and most probably after dissolution,
too, and this is the most likely binding for [CuL]+ of 1a and
[CuLH]+ of 1c in the equilibrium systems as well (1). (It should
be noted that, for simplicity, the coordinated solvent molecules
are omitted here and later.) The anisotropic ESR parameters
(Table 5) are in good agreement with similar data for the
Table 5 ESR parametersa at 77 K for the Cu(II) complexesb of 1a–1c in 80% (w/w) DMSO/water
c
c
%
Ligand
Complex
gxx
gyy
gzz
g0
Axx/G
Ayy/G
Azz/G
A0 /G
Solvent
1a
Cu2+
2.0766(2)
2.0894(3)
2.0585(1)
ESR-inactive
ESR-inactive
ESR-inactive
2.0587(2)
2.0498(3)
2.0788(5)
2.0766(2)
2.0523(3)
2.0555(2)
2.4060(5)
2.3490(7)
2.3148(7)
2.186
2.164
2.143
0.2(4)
2.6(5)
4.9(3)
0.2(9)
7.5(6)
6.9(7)
112.4(3)
124.8(6)
135.7(4)
37.6
40.0
49.2
[CuL]+
[CuL(OH)]
[Cu2L2(OH)4]2-
[Cu2L2]2+
[Cu2L2H-2]
[CuLH-2]-
1b
1c
2.0483(4)
2.0347(1)
2.0510(3)
2.0459(2)
2.0435(6)
2.2740(3)
2.2467(4)
2.3483(5)
2.3041(2)
2.2901(3)
2.127
2.110
2.156
2.140
2.134
5.9(9)
30.2(6)
4.3(3)
3.4(3)
4.9(1)
7.4 (2)
172.3(4)
187.0(3)
130.0(4)
148.1(5)
156.4(3)
69.5
72.4
44.9
53.2
58.7
48.5
51.5
25.8(7)
1.25(1)
6.6(2)
[CuLH]+
[CuLH(OH)]
2.0693(4)
2.0648(6)
[CuLH(OH)2]-
12.4(3)
a The confidence intervals of the parameters regarding the last digit(s) are given in parentheses. The hyperfine coupling constants are given in G (10-4
T) units, and refer to the isotope 63Cu. b The solvent molecules bound in the first coordination sphere are omitted. c Average of the principal values of the
g- and A-tensors, respectively.
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mono-ligand copper(II) complexes of the bidentate Schiff-bases
formed from 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one
with 3-formyl-6-methyl-chromone.21 At pH > 6, the proton loss of
the coordinated water molecules occurs, resulting in the formation
of [CuL(OH)] and [Cu2L2(OH)4]2- with 1a, and [CuLH(OH)]
and [CuLH(OH)2]- with 1c (as above). At pH ª 9.2 (which
carried out for the visualization of several possible structures.
If the first ligand is bound by its N and phenolate O- atoms
to the first Cu(II), its phenolic OH group can occupy the third
equatorial site of another Cu(II), and vice versa (5), offering a
good possibility for a strong exchange interaction between the
copper(II) centres. For [Cu2L2H-2], both phenolic OH groups are
deprotonated. The former type of coordination can be formed as
well, with not phenolic but phenolate bridges, however (6). The
outstanding stability of the former two complexes of 1b (Table 4)
can be explained by the formation of a third strong equatorial
bond with each metal ion in the dimeric structure. If the second
OH group is in the meta position to the first one, the formation of
this structure is sterically hindered.
For 1b, the well-resolved, regular anisotropic spectrum of
[CuLH-2]- can be described as the superposition of two component
curves, suggesting an isomeric equilibrium in solution. Both
component spectra (Fig. 6) are quite different from the spectra
of the complexes of the other ligands: they can be characterized
by much lower g and higher A values, and their narrow lines can be
attributed to the lack of a non-resolved N splitting; in other words,
these spectra indicate merely O donors in the equatorial plane.
The formula [CuLH-1(OH)] could reflect on the structure of this
species: most probably, here the catecholate coordination occurs,
together with a hydroxide ion in the equatorial or axial position.
For the major isomer, lower g values (Table 5) correspond to the
equatorial ligation of the hydroxide ion (7).
can be regarded as neutral in this solvent mixture with pKw
=
18.4) and in slightly alkaline solution (which corresponds to
the “physiological” pH in living organisms), namely these mixed
hydroxido complexes are present under equilibrium conditions.
Accordingly, these complexes which may play a role in the
biological effects of the ligands if Cu(II) is present. The decrease
of g and increase of A (Table 5) indicate even stronger equatorial
ligand field as the number of hydroxide ions in the complex in-
creases, supporting that hydroxide ions enter the equatorial plane
(determined by the chelating atoms) rather then apical positions
in the monomeric species (2,3). A moderate rhombic splitting of g
reflects on some distortion of the coordination polyhedra. For
1a, in the ESR-inactive [Cu2L2(OH)4]2-, the strong interaction
between the Cu(II) ions is most probably mediated by two,
hydroxido bridges, while two further hydroxide ions occupy apical
positions (4).
Ligand 1b contains three possible donor groups on adjacent
carbon atoms of the aromatic ring, however, only two of them
can be bound to the same Cu(II) simultaneously for steric
reasons. In principle, two coordination modes are possible for
this molecule: either the Schiff-base-type ligation by the N and
the neighbouring phenolate O-, or the catecholate-like binding
through two phenolate O- atoms. The third group, not involved
in chelation, is free to be bound to another metal ion. For
[Cu2L2]2+, only one of the two phenolic OH groups of the ligand
is deprotonated and coordinated to Cu(II). This follows from the
composition of the complex and the fact that the ligand does
not have any atom on it which could bind the second proton if
both phenolic OH were deprotonated (see the pH-potentiometric
results). That is, a Schiff-base-like coordination is probable also
in this case. The question arises, how the strong interaction
between the Cu(II) ions, leading to spin-pairing, can be formed.
Assuming that the non-deprotonated phenolic OH groups can
also coordinate to metal ions, Hyperchem MM+ calculations were
Fig. 6 Calculated spectra for the isomers of the complex [CuLH–2]- of
ligand 1b.
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Table 6 ESR parametersa for the bis complexes 2a, 2b and 2c in 80% (w/w) DMSO/water at 77 K
b
Complex
gxx
gyy
gzz
g0
Axx/G
Ayy/G
Azz/G
A0b/G
%
2a
2.0877(6)
2.0847(5)
2.0650(2)
2.0493(3)
2.0677(3)
2.0351(4)
2.0486(5)
2.0650(2)
2.0783(4)
2.0431(3)
2.2500(3)
2.2591(8)
2.3102(8)
2.3139(5)
2.2473(4)
2.124
2.131
2.147
2.147
2.119
4.2(2)
1.1(3)
0.4(10)
7.0(3)
1.4(8)
5.1 (2)
23.5(2)
0.4(10)
2.1((4)
14.4(5)
155.9(3)
158.0(8)
149.0(9)
147.3(5)
155.2(6)
55.1
60.9
49.9
52.1
57.0
100
64
36
100
100
2bc
2bd
2c
a The confidence intervals of the parameters regarding the last digit(s) are given in parentheses. The hyperfine coupling constants are given in G (10-4
T) units, and refer to the isotope 63Cu. b Average of the principal values of the g- and A-tensors, respectively. c Immediately after dissolution. d After 4 h.
ESR spectra of the bis complexes
narrow lines indicate oxygen coordination in the equatorial plane.
Most probably, [CuL2H-2]2- with bis(catecholate) coordination is
formed. Then slow spectral changes indicate further structural
changes.
The bis complexes are thermodynamically unstable when dissolved
in 80% (w/w) DMSO/water as they are not formed in the solutions
of the ligands and Cu(II) under similar conditions. Their ESR
spectra (Fig. 7) are quite different from all other curves found
in the corresponding equilibrium systems. The anisotropic ESR
parameters for the bis complexes are summarized in Table 6.
The gzz and g0 values are much lower than for the mono-ligand
complexes,21 Table 5), indicating a stronger ligand field and
supporting the equatorial coordination of two N and two O
donors, in accordance with the Schiff-base-type coordination
of two ligands in the complexes studied. The N superhyperfine
coupling constants give less structural information, since they can
be determined with high uncertainty, as a consequence of the
large linewidths. The rhombic splitting of g (|gxx - gyy| > 0.3)
indicates the distortion of the coordination polyhedra. For 2a and
2c, the solutions are nearly neutral as expected (pH = 8.6 and
8.7, respectively), and there is no significant pH change within
a week.
Conclusions
This paper investigated the possible therapeutic activity of a new
series of secondary amines derived from Schiff base ligands. In
addition, the antimicrobial and anticancer activity of Schiff bases
and their copper(II) complexes were also investigated. Whilst the
secondary amines showed little anti-Candida activity they did
exhibit anticancer activity comparable to a commercial anticancer
drug. Their lack of anti-Candida activity was attributed to the
lack of imine functionality present, which seemed to be necessary
for anti-Candida activity. None of the compounds tested showed
significant activity against microbial species.
The solution behaviour of the ligands and their copper(II)
complexes could only be examined for those complexes whose sol-
ubility was reasonable in DMSO:water mixtures. In a comparison
with the previously studied salicaldehyde complexes of copper(II),
it was found that the Schiff-base ligands form more stable
complexes in solution than salicylaldehyde with the complexes
of 1b being the most stable. A previous study had identified the
potent anti-Candida activity of the copper(II) complex of 1b,7
where a bis complex had been characterised in the solid state.
Similarly to the crystalline state, the Schiff-base-like coordination
of the ligands to Cu(II) through the N and the phenolate O- is
predominant in solution equilibrium systems, too, where, however,
only mono complexes can be identified. The free coordination sites
are occupied by solvent molecules at low pH, while in neutral and
alkaline solution, mixed hydroxido complexes are formed. The
stabilities and coordination modes of the corresponding Cu(II)
complexes of various ligands do not differ significantly, with one
exception. When the ligand also contains another OH group, in the
ortho position relative to the first one (1b), this occupies the third
equatorial site of the metal ion of another monomeric unit, and
vice versa, forming a highly stable dimeric complex in the acidic
pH region. At nearly neutral pH, these bridging OH groups of the
dimer are deprotonated. In alkaline solution, the binuclear species
are replaced by a mixed hydroxido complex with catecholate
coordination, which is also significantly more stable than the other
mixed hydroxido complexes with Schiff-base binding mode for the
other ligands. For the bis complex of this ligand, the Schiff-base-
type coordination is also replaced by a catecholate-like ligation,
immediately when dissolved. An extension of this study to identify
speciation of the complexes in living cells is currently underway.
Fig. 7 ESR spectra of the copper(II) complexes dissolved in 80% (w/w)
DMSO/water (black: experimental, gray: calculated curves); for 2b, the
upper curve was recorded immediately after dissolution, while the lower
spectrum was obtained 4 h later.
The complex 2b undergoes a significant structural change
when it is dissolved. Immediately after dissolution, its spectrum
is reminiscent of the curves of 2a and 2c, however, a new
component is also present, which becomes predominant within
4 h (Fig. 7). This spectral change is accompanied with a significant
pH decrease: from 8.5 to 6.8 for the solution of 2b after ageing for
4 h. In the first step, protons are released (pH decreases), and the
spectrum alters significantly: the changes in the parameters and the
10864 | Dalton Trans., 2010, 39, 10854–10865
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2008, 102, 101–109; R. S´ıposˇ, T. Szabo´-Pla´nka, A. Rockenbauer, N. V.
Acknowledgements
ˇ
Nagy, J. Sima, M. Meln´ık and I. Nagypa´l, J. Phys. Chem. A, 2008, 112,
10280–10286.
This research was supported by the Technological Sector Research
Programme, Strand 1, under the European Social Fund. D.K.
gratefully acknowledges the EMBARK fellowship for additional
funding. The financial support of the Hungarian Scientific Re-
search Fund OTKA (Grants K72781 and F67581) is gratefully
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