New copper(II) niflumate complexes with N-donor ligands: Synthesis, characterization and evaluation of anticancer potential against human cell lines

Article abstract of DOI:10.1016/j.ica.2019.01.020

Five new copper(II) niflumate complexes, [Cu(en)₂(nif)₂], 1, [Cu(unsym-dmen)(nif)₂], 2, [Cu(pn)₂(nif)₂][Cu(pn)₂(H₂O)₂](nif)₂ 3, [Cu(β-pic)₂(nif)<

Full text of DOI:10.1016/j.ica.2019.01.020

Accepted Manuscript
Research paper
New copper(II) niflumate complexes with N-donor ligands: synthesis, charac-
terization and evaluation of anticancer potential against human cell lines
Santosh Kumar, Raj Pal Sharma, Paloth Venugopalan, Valeria Ferretti, Michel
Tarpin, Stéphanie Sayen, Emmanuel Guillon
PII:
S0020-1693(18)31704-3
https://doi.org/10.1016/j.ica.2019.01.020
ICA 18743
DOI:
Reference:
Inorganica Chimica Acta
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Accepted Date:
7 November 2018
16 January 2019
17 January 2019
Please cite this article as: S. Kumar, R. Pal Sharma, P. Venugopalan, V. Ferretti, M. Tarpin, S. Sayen, E. Guillon,
New copper(II) niflumate complexes with N-donor ligands: synthesis, characterization and evaluation of anticancer
potential against human cell lines, Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.01.020
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New copper(II) niflumate complexes with N-donor ligands: synthesis, characterization and
evaluation of anticancer potential against human cell lines
Santosh Kumar,a Raj Pal Sharma,a,* Paloth Venugopalan,a Valeria Ferretti,b,* Michel Tarpin,c
Stéphanie Sayen,d Emmanuel Guillond,*
aDepartment of Chemistry and center of advanced studies, Panjab University, Chandigarh,
India-160014
bDepartment of Chemical and Pharmaceutical Sciences and Center for Structural
Diffractometry, University of Ferrara, via Fossato di Mortara 17-27, 1-44121, Ferrara, Italy
cLaboratoire Signalisation Cellulaire et Récepteurs Matriciels (SiRMa, UMR CNRS 7369
MEDyC), Université de Reims Champagne-Ardenne, F-51687 Reims Cedex 2, France.
dInstitut de Chimie Moléculaire de Reims (ICMR, UMR CNRS 7312), Groupe Chimie de
Coordination, Université de Reims Champagne-Ardenne, F-51687 Reims Cedex 2, France.
Email: rpsharmapu@yahoo.co.in (RPS) ; frt@unife.it (VF); emmanuel.guillon@univ-reims.fr
(EG)
1

Abstract
Five new copper(II) niflumate complexes, [Cu(en)2(nif)2], 1, [Cu(unsym-dmen)(nif)2], 2,
[Cu(pn)2(nif)2][Cu(pn)2(H2O)2](nif)2 3, [Cu(-pic)2(nif)2], 4, [Cu(-pic)2(nif)2] 5 (where en
=ethylenediamine, pn = propan-1,3-diamine, unsym-dmen = unsymmetrical 1,1-dimethyl-
ethylenediamine, -pic = 3-methylpyridine, -pic = 4-methylpyridine, nif = niflumate) have been
synthesized by using appropriate starting materials and methanol-water (4:1 v/v) as solvent. All
the above synthesized complexes 1-5 have been characterized by spectroscopic methods (UV-
Vis, FT-IR, and EPR). The structures of complexes 1, 3 and 5 have been unambiguously
determined by single crystal X-ray structure determination which clearly revealed that these
complexes are mononuclear complexes. The structure of complex 4 has been optimized by DFT
calculations. In all complexes 1-5, niflumate shows direct coordination to central copper(II) ion
leading to covalent character of all complexes, although the complex 3 also contains cationic
moiety [Cu(pn)2(nif)2]2+ and two nifumate ions. The ability of all the complexes to show
cytotoxicity against human cancer cell lines has also been evaluated.
Keywords: copper(II) complexes; niflumic acid; NSAIDs; cytotoxicity; cancer cell lines
2

1. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are such an important class of drugs,
capable of alleviating pain, inflammation and fever in both humans and animals (with tolerable
side effects), that they are available from a chemist shop without a medical prescription. What is
more remarkable about them is that they can also show significant antitumor activities either by
themselves (cancer cell death by apoptosis) or through a mechanism involving free radicals or in
synergistic combination with other antitumor agents [1-4]. Even though much diversification
exists within NSAIDs and sub-classifications are possible (e.g., salicylates, oxicams, furanones,
fenamates etc.), a commonality that can be traced among most of them is the presence of an
arylcarboxylate group [4-6]. A typical and important example is niflumic acid, (2-{[3-
(trifluoromethyl)phenyl]amino}-3-pyridine-carboxylic acid, Hnif), which acts like an enzyme
inhibitor against the prostaglandin-producing cyclooxygenase-1 (COX-1) and cyclooxygenase-2
(COX-2) enzymes [6-9]. Other very commonly used NSAIDs against COX-1 and COX-2 which
possess an arylcarboxylate group are shown in Scheme 1.
CH3
O
CH3
COOH
COOH
COOH
Ph
Ph
F
Ibuprofen
Ketoprofen
Flurbiprofen
COOH
Cl
HO
H
N
N
H3CO
F3C
N
H
Cl
COOH
COOH
Niflumic acid
Ferulic acid
Diclofenac acid
Scheme 1. Structural formulae of some commonly used NSAIDs [4, 6]
Besides being an important class of NSAIDs, arylcarboxylates are also equally important
class of ligands in bioinorganic chemistry that exhibit a wide range of coordination modes and
associated geometries. Once deprotonated, the arylcarboxylate anion such as niflumate (nif) can
3

have four lone pairs of electrons available for metal binding. Indeed, metal coordinated
arylcarboxylate complexes (i.e., M-arylcarboxylates) are highly investigated in bio-inorganic
chemistry as they show wide structural varieties (with different geometries) and important
biological properties [10-12].
“Efficacy enhancement” with “minimal side effects” is always the leading impetus for a
medicinal/bioinorganic chemist working in the NSAID area and two approaches that have
become highly successful are (a) trying to alter the coordination environment by judicially
choosing/replacing a metal ion that is capable of coordinating with a chosen drug, [13-15] (b)
using ancillary ligands (e.g. triphenylphosphine (tpp), phenanthroline, bipyridyl, pyrazine etc.,)
that can also penetrate/associate with the existing metal ion coordination environment in the
complex [13, 14, 16-18]. It is also possible to use a combination of both the approaches
mentioned above. In this regard, copper (mainly in the +2 oxidation state) becomes an important
and judicious choice as it is a biologically essential element in most organisms and its
absorption, homeostasis and excretion mechanisms are well linked and properly maintained
along the evolutionary lines of biological systems [19-21]. Hence, ingestion of copper to living
cells, even if through coordinated NSAIDs [22-23] should evoke a minimal problem with regard
to side effects of metal poisoning. Moreover, it has been well established that complexation with
metal ions can alter the efficacy of a drug [24-26] and it would be rewarding to investigate the
copper(II) complexes of an important NSAID, niflumic acid. It is a well-known fact that N-donor
compounds, as ancillary ligands, can alter the properties and structures of metal-
arylcarboxylates. Nitrogen donor ligands can also enhance the biological activity of transition
metal complexes by altering coordination modes of other co-ligands towards the metal center,
e.g. the cytotoxicity of monomeric [Cu(asp)2(py)2] towards various tumor cell lines is higher
4

than that of the dimeric [Cu2(asp)4] complex [27], where py = pyridine. In light of the above
discussion we found it pertinent to investigate the spectroscopic, structural and biological
activities of the niflumate anion (nif) after its complexation with copper(II) in the presence of a
variety of N-donor ligands as shown in Scheme 2. The selection of the ancillary ligands, that
either belong to a category of flexible aliphatic ligands such as ethylenediamine (en), propan-1,3-
diamine (pn), 1,1-dimethylethylenediamine (unsym-dmen)) or that belong to class of resonance-
stabilized aromatic ligands such as -methylpyridine (-pic), -methylpyridine (-pic), was
based on the fact that they can impart structural variations due to different coordination modes of
the arycaboxylate group and thereby affect the biological properties.
H2N
N
H2N
NH2
NH2
NH2
(a)
(b)
(c)
COOH
H
F3C
N
N
N
N
(d)
(e)
(f)
Scheme 2: Ligands used in this work (a) ethylenediamine (en), (b) propan-1,3-diamine (pn), (c)
1,1-dimethylethylenediamine (unsym-dmen), (d) -picoline (-pic), (e) -picoline (-pic), (f)
niflumic acid (Hnif)
In the literature, a few papers on X-ray structural studies of metal-niflumate complexes; Zn(II)
[28], Cu(II) (one monomeric, three dinuclear, one polymeric), [29]] Mn(II) [30] , Co(II) [31],
and [Ag(I)] [32] have been reported. Hence, in order to study the coordination chemistry of
copper(II) niflumate with aliphatic and aromatic N-donor ligands as an extension of our synthetic
methodology [33], we report herein the synthesis and elaborate characterization of five new
5

copper(II) niflumate complexes, i.e. [Cu(en)2(nif)2], 1, [Cu(unsym-dmen)(nif)2], 2 [Cu(pn)2(nif)2]
[Cu(pn)2(H2O)2](nif)2, 3, [Cu(-pic)2(nif)2], 4, [Cu(-pic)2(nif)2] 5, besides their cytotoxic
evaluation against various human cancer cell lines for a potential application as anticancer
agents. The findings that emerge from these studies can augment the knowledge regarding the
synthetic, spectroscopic and structural aspects of Cu(II) arylcarboxylates including NSAIDs, an
area to which we have embarked upon for quite some time [34,35]. It is worth mentioning that
this work is based upon a methodology concerning the synthesis and characterization of
copper(II) arylcarboxylate complexes with N- donor ligands that has been developed over two
decades and presents salient features, such as: (i) the materials are cheap and readily available;
(ii) the synthetic method is simple, convenient and proceeds without any sophistication at room
temperature; (iii) the obtained products are crystalline, pure and with nearly quantitative yield;
(iv) usually no recrystallization is needed for further purification; (v) good quality crystals are
readily obtained for single crystal X-ray structural analysis.
2. Experimental
2.1 Materials and methods
All chemicals and solvents were reagent grade and were used as purchased without any further
purification. Elemental analysis was performed using an automatic Perkin Elmer 2400 CHN
element analyzer and copper was determined by standard literature methods [36]. Fourier
transform infrared spectra (FT-IR) were recorded (neat) on PERKIN ELMER SPECTRUM RX
FT-IR system. The UV-vis spectra of the complexes were recorded on a Hitachi U-2001 dual
beam spectrophotometer using methanol-water (4:1 v/v) mixture as solvent. The anisotropic X-
band (9.43 GHz) EPR spectra of frozen CHCl3 solutions were recorded at 110 K using a Bruker
6

ESP 300 spectrophotometer. The EPR spectra were referenced to 2,2-diphenyl-1-picrylhydrazyl
(DPPH) (g=2.0037). All spectra were recorded using 100 kHz frequency modulation, 21.186 G
amplitude modulation, and microwave power of 6 mW.
2.2 Synthesis of [Cu(en)2(nif)2].2H2O, 1
0.50 g CuSO4.5H2O (2 mmol) was dissolved in 10 mL of distilled water and sodium niflumate
(prepared in situ by mixing aqueous sodium hydroxide (0.16 g, 4 mmol) with niflumic acid
(1.12 g, 4 mmol)) which was also dissolved in minimum amount of water. On mixing the two
solutions, a light green precipitate of hydrated copper(II) niflumate resulted immediately which
was filtered through a fine filter paper, washed with water followed by methanol and dried at
room temperature (yield 90%). 0.5 g hydrated copper(II) niflumate was suspended in 25mL
methanol-water (4:1 v/v) and ethylenediamine (en) was added slowly with stirring until a clear
violet solution appeared. When the reaction mixture was allowed to evaporate slowly at room
temperature, violet crystals appeared after a few days, which were separated from the mother
liquor, washed with water and dried in air (yield 90%). Complex 1 is soluble in methanol,
acetone but insoluble in water and decomposes at 190 oC. FT-IR (neat, cm-1): 3420(m), 3256(m),
3157(s), 2950(m), 1574(s), 1489(m), 1367(s), 1309(s), 1251(m), 1032(s), 941(w), 784(s), 694(s),
541(s); ΔνCOO = νasym(COO) - νsym(COO) = 207 cm-1 . UV-Vis (methanol-water), λmax(nm) (ε, in M-
1cm-1): .575 (58). Anal. Cal. for C30H36N8O6F6Cu (MW = 782.21): C, 46.02; H, 4.60, N, 14.32;
Cu, 8.18 %; found: C, 46.55; H, 4.32; N, 13.98; Cu, 8.37 %.2.3 Synthesis of [Cu(unsym-
dmen)2(nif)2], 2
Complex 2 was prepared in the similar manner as complex 1 by using 1,1-
dimethylethylenediamine (unsym-dmen) instead of ethylenediamine (en). When the reaction
mixture was allowed to evaporate slowly at room temperature, violet coloured crystals appeared
7

after a few days, which were separated from the mother liquor washed with methanol and dried
in air (yield 85%). Complex 2 is also partially soluble in water, insoluble in methanol, acetone
o
and decomposes at 201 C. FT-IR (neat, cm-1): 3222(s), 3063(m), 2890(m), 2820(w), 1578(s),
1384(s), 1323(s), 1254(m), 1114(m), 899(w), 762(s), 521(m) ; ΔνCOO = νasym(COO) - νsym(COO)
=
194 cm-1 . UV-Vis (methanol-water), λmax(nm) (ε, in M-1cm-1): 560 (162). Anal. Cal. for
C34H40N8O4F6Cu (MW = 801.50): C, 50.90; H, 4.99, N, 13.97; Cu, 7.92 %; found: C, 51.06; H,
5.13; N, 13.68; Cu, 7.82 %.
2.4 Synthesis of [Cu(pn)2(nif)2].[Cu(pn)2(H2O)2](nif)2, 3
Complex 3 was synthesized in a similar manner as complex 1 by adding propane-1,3-diamine
(pn) instead of ethylenediamine (en) till a clear blue color solution was obtained. When the
reaction mixture was allowed to evaporate slowly at room temperature, blue crystals appeared
after four days, which were separated from the mother liquor washed with methanol and dried in
air (yield 88%). Complex 3 is also soluble in water, insoluble in methanol and decomposes at
o
182 C. FT-IR (neat, cm-1): 3480(b), 3277(s), 2938(m), 1579(s), 1383(s), 1116 (s), 1202(s),
941(s), 775(s), 670(m), 521(m) 456(w); ΔνCOO = νasym(COO) - νsym(COO) = 197 cm-1 . UV-Vis
(methanol-water), λmax(nm) (ε, in M-1cm-1): 563 (188). Anal. Cal. for (C64H76N16O10F12Cu2)
(MW = 1584.48): C, 48.47; H, 4.79, N, 14.13; Cu, 8.08 %; found: C, 48.55; H, 4.58; N, 14.45;
Cu, 8.23 %.
2.5 Synthesis of [Cu(-pic)2(nif)2], 4
Complex 4 was synthesized in a similar manner as complex 1 by adding -picoline (-pic) in
place of ethylenediamine (en) until a clear blue color solution was obtained. When the reaction
mixture solution was allowed to evaporate slowly at room temperature, blue crystals appeared
after three days, which were separated from the mother liquor, washed with water and dried in
8

air (yield 85%). Complex 4 is insoluble in water and soluble in methanol and decomposes at 183
oC. FT-IR (neat, cm-1): 3260(m), 2980(m), 1587(s), 1518(s), 1443(m), 1336(s), 1065(s), 775(s),
660(s), 534(s), 458(m); ΔνCOO = νasym(COO). - νsym(COO) = 251 cm-1 . UV-Vis (methanol-water),
λmax(nm) (ε, in M-1cm-1): 728 (108). Anal. Cal. for C38H30N6O4F6Cu (MW = 812.22): C, 56.14;
H, 3.69, N, 10.34; Cu, 7.87 %; found: C, 56.33; H, 3.55; N, 10.26; Cu, 7.53 %.
2.6 Synthesis of [Cu(-pic)2(nif)2], 5
Complex 5 was synthesized in a similar manner as complex 1 by adding -picoline (-pic) in
place of ethylenediamine until a clear royal blue color solution was obtained. When the reaction
mixture solution was allowed to evaporate slowly at room temperature, royal blue crystals
appeared after a few days, which were separated from the mother liquor washed with water and
dried in air (yield 92%). Complex 5 is also insoluble in water and soluble in methanol, other
o
organic solvents and decomposes at 210 C. FT-IR (neat, cm-1): 3069(s), 1595(s), 1346(s),
1445(3), 1066(m), 761(s), 547(m), 492(s); ΔνCOO = νasym(COO) - νsym(COO) = 222 cm-1 . UV-Vis
(methanol-water), λmax(nm) (ε, in M-1cm-1): 735 (137). Anal. Cal. for C38H30N6O4F6Cu (MW =
812.22): C, 56.14; H, 3.69, N, 10.34; Cu, 7.87 %; found: C, 55.97; H, 3.76; N, 10.17; Cu, 7.93
%.
2.7 Biological tests
Cell Cultures. Two cell types were studied. The HT-144 and SKMEL-28 cell lines derived from
human melanoma and were obtained from the American Tissue Culture Collection (ATCC).
They were cultured in DMEM, Mc Coy’s 5a modified or MEM culture medium (pH 7.4,
controlled by the presence of the pH indicator (phenol red) in the medium supplemented with 10
% FBS.
9

Cell viability assay. Initial stock solutions of complexes were prepared in dimethyl sulfoxide
(DMSO) at a concentration of 10-1 M and used to achieve the several dilutions. The effects of
complexes on cell viability at 24h were measured using an MTT tetrazolium salt colorimetric
assay (Sigma-Aldrich, Saint-Louis, Missouri, United States) according to the manufacturer’s
instructions. Briefly, the cells (15000/well) were incubated in three replicates in a 96-well plate
in the presence of various concentrations of complexes (0, 6.25, 12.5, 25, 50, 100, 200 µM in the
wells). After 24 hours of treatments, cells were incubated for three hours after addition of 20%
MTT, then medium was removed and DMSO was added to dissolve formazan. A colorimetric
assay was realized at 560nm using a plate reader Tecan F200 Pro (Tecan, Lyon, France). The
50% inhibitory concentration (IC50) for each complex was defined as the concentration
producing 50% decrease in cell growth.
2.8 Statistical Analysis
The data were expressed as the mean _ SD of 3 independent experiments. Each experiment was
performed in triplicate. The significance of differences was established with the Student’s t-test.
2.9 X-ray Crystallography
Single-crystal diffraction data for complexes 1, 3 and 5 were collected at 295 K on a Nonius
Kappa diffractometer equipped with a CCD detector with graphite-monochromated Mo-Kα
radiation (λ = 0.71069 Ǻ ). Intensities were corrected for Lorentz, polarization and absorption
effects [37]. The low quality/smallness of the crystals of 1 and 5 did not allow a high data
completeness fulfilment. The structures were solved by direct methods with the SIR97 program
[38] and refinements were performed on F2 by full matrix least-squares methods with all non-H
atoms anisotropic, apart from the disordered O1w atom in 3 which was refined isotropically. In
complex 5, the fluorine atoms of the CF3 group of the niflumic ligand were found to be
10

disordered over two equivalent positions. In all structures, the C-H hydrogens were included on
calculated positions, riding on their carrier atoms. All other N/O-H hydrogen atoms were located
in the difference-Fourier map and refined isotropically, apart from those of the water molecule
coordinated to Cu2 in complex 3 (not included in the refinement).
Diffraction data for complex 4 were collected as well; in spite of many efforts, however, it was
not possible to refine the structure to a reasonable R factor value. For this reason the geometry of
the complex was optimized via DFT calculations (vide infra). All calculations were performed
using SHELXL2014/7 [39] implemented in the WinGX system of programs [40]. Experimental
details are given in Table S1 (Supplementary data).
Crystallographic data for the structural analysis of the three new compounds have been deposited
at the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge, CB2 1EZ, UK, and
are available free of charge from the Director on request quoting the deposition number CCDC
1824661-1824663 for complexes 1, 5 and 3 respectively.
2.10 DFT calculations
The geometry of the complex [Cu(-pic)2(nif)2] 4 was optimized in vacuum without any
symmetry constraints, starting from the crystal structure geometry. Calculations were carried out
with the Gaussian09 suite of programs [41] employing the B3LYP [42] functional in
combination with a LANL2DZ [43] basis set.
3. Results and Discussion
3.1 Synthesis
The hydrated copper(II) niflumate was obtained by reacting copper sulfate pentahydrate
with the sodium salt of niflumic acid as shown in Scheme 3 (eq. (i)). The precipitated product
11

was then suspended in methanol-water mixture (4:1, v/v) followed by the addition of different
nitrogen-donor ligands (en, pn, unsym-dmen, -picoline and -picoline) with continuous stirring
until a clear blue/violet solution was observed in each case. Upon slow evaporation of the
respective resultant reaction mixtures at room temperature, five new copper(II) niflumate
complexes 1-5 were isolated in good yields.
H2O, RT
[i]
.
+Na SO
[Cu2(nif )4(H2O)2]
CuSO4 5H2O+2HNif + 2NaOH
2
4
en (excess)
[Cu(en)2(nif )2].2H2O
[ii(a)]
1
unsym-dmen(excess)
[ii(b)]
[Cu(unsym-dmen)2(nif )2]
2
pn (excess)
-pic(excess)
 -pic(excess)
MeOH:H2O,
RT
[ii(c)]
[Cu2(nif )4(H2O)2]
[Cu(pn)2(nif )2].[Cu(pn)2(H2O)2](nif )2
3
[Cu(-pic)2(nif )2]
[ii(d)]
[ii(e)]
4
[Cu( -pic)2(nif )2]
COOH
5
H
N
en= ethylenediamine
pn= propan-1,3-diamine,
F3C
where Hnif =
N
unsym-dmen = 1,1-dimethyl-ethylenediamine
-pic= 3-methylpyridine
-pic= 4-methylpyridine
Scheme 3. Schematic representation of the synthesis of complexes 1-5
The composition of each complex has been confirmed by elemental analyses and the exact
structure of complexes 1, 3 and 5 was confirmed by X-ray structure determinations.
3.2 X-Ray crystal structures of complexes 1, 3 and 5
The ORTEPIII views [44] of complexes 1, 3 and 5 are shown in Figs. 1, 2 and 3, respectively. In
all structures, the central copper atom lies on a symmetry centre. In complex 1, the coordination
geometry is elongated octahedral, the Cu atom being coordinated to two chelating en ligands and
two monodentate niflumate ligands in apical positions. For each complex, outside the first
coordination sphere, there are two co-crystallized water molecules. The five membered ring Cu1-
12

N1-C1-C2-N2 adopts a twisted conformation according to the Cremer and Pople analysis
(puckering amplitude q2 = 0.44, pseudorotation phase angle φ2 = 122.9°) [45]. The Cu-N
distances, reported in Table 1, are perfectly in line with those found in Cu(en) structures, for
which the mean Cu-N distance is 2.01(3) Å (571 structures in CSD).
The structure of complex 3 is characterized by the presence of two different Cu complexes, as
shown in Fig. 2. In the first one (Cu1 complex) the coordination is similar to that found in
complex 1, with the niflumate ligands in apical positions of a distorted octahedron; conversely,
the second copper atom (Cu2) is bound with a distorted octahedral geometry to two pn chelating
ligands and two water molecules to give a cationic species, while the niflumate anionic moieties
are located outside the first coordination sphere. Both the Cu1-N1-C1-C2-C3-N2 and Cu2-N5-
C17-C18-C19-N6 six-membered rings adopt a chair conformation, with a total puckering
amplitude of 0.609 and 0.672 and spherical polar angles of 7.42 ° and 9.13°, respectively.
13

Table 1. Selected bond distances and angles (Å, °) for complexes 1, 3 and 5.
Complex 1
Cu1 - O1
Cu1 - N2
2.606(2)
2.010(2)
Cu1 - N1
2.008(2)
O1 - Cu1 - N1
O1 - Cu1 - N2
91.97(7)
88.09(7)
N1 - Cu1 - N2
N1 - Cu1 - N2i
84.66(9)
95.34(9)
Complex 3
Cu1 - N1
Cu1 - O1
Cu2- N6
2.018(2)
2.685(2)
2.011(2)
Cu1- N2
Cu2- N5
Cu2- O1W
2.011(3)
2.016(2)
2.500(8)
N1 - Cu1 - N2
O1 - Cu1- N2
O1 - Cu1- N1
N5 - Cu2 - N6
N6 - Cu2 - O1W
O1w - Cu2 - N6iii
89.6(1)
101.8(1)
94.0(1)
86.6(1)
89.6(2)
90.4(2)
N1 - Cu1 - N2ii
O1 - Cu1- N1ii
O1 - Cu1 - N2ii
N5 - Cu2 - O1W
N5 - Cu2 - N6iii
O1w - Cu2 - N5iii
90.3(1)
86.0(1)
78.2(1)
85.4(2)
93.3(1)
94.6(1)
Complex 5
Cu1 - N1
Cu1 - O1
2.051(2)
1.942(2)
Cu1 - O2
2.664(2)
N1 - Cu1 - O1
N1 - Cu1 - O2
90.21(8)
89.11(8)
O1- Cu1- O2
O1- Cu1- O2iv
54.51(8)
125.49(8)
Equivalent positions: (i) 1-x,-y,1-z; (ii) 1-x,1-y,1-z; (iii) -x,1-y,1-z; (iv) -x,-y,1-z
14

Table 2. Structural parameters of hydrogen bonds (Å, °) for complexes 1, 3 and 5.
D-H…A
D-H
D…A
H…A
D-H…A
Complex 1
N1-H ...O2
N4-H ...O2
0.84(3)
0.89(3)
0.76(4)
0.90(3)
0.93
0.79(4)
0.93(3)
0.78(4)
3.053(3)
2.672(3)
2.799(3)
3.042(3)
3.507(3)
3.121(3)
3.093(3)
2.828(3)
2.26(3)
1.92(3)
2.05(4)
2.24(3)
2.62
2.33(3)
2.26(3)
2.06(4)
158(2)
141(2)
172(4)
148(3)
157
173(3)
147(2)
175(4)
O1W-H...O2
N2-H ...O1Wi
C12-H...F3ii
N2-H ...O1Wiii
N1-H ...O1Wiv
O1W-H...O1v
Equivalent positions: (i) 3/2-x,y-1/2,z; ( ii) x+1/2,y,3/2-z; (iii) 1-x,-y,1-z; (iv) x-1/2,1/2-y,1-z;
(v) 2-x,-y,1-z
Complex 3
N3-H...O1
N7-H...O3
C3-H...N4i
N5-H...O2ii
N1-H...O4iii
N1-H...O2iv
N6-H...O2iv
N2-H...O4v
N5-H...O4vi
N6-H...O3vi
0.70(3)
0.76(3)
0.97
0.85(4)
0.91(4)
0.90(4)
0.85(4)
0.85(5)
0.76(4)
0.96(4)
2.656(3)
2.660(3)
3.386(5)
2.930(3)
3.081(3)
2.894(4)
2.887(3)
3.032(4)
2.964(4)
3.005(3)
2.03(3)
1.99(3)
2.50
2.09(4)
2.21(4)
2.07(4)
2.05(4)
2.19(5)
2.24(4)
2.05(4)
150(3)
149(3)
151
166(3)
161(4)
150(3)
167(4)
172(5)
167(4)
163(3)
Equivalent positions: (i) x,1/2-y,z+1/2; (ii) x-1,y,z; (iii) x,1/2-y,z-1/2; (iv) 1-x,1-y,-z-1;
(v) 1-x,y+1/2,-z-1/2; (vi) -x,y+1/2,-z-1/2
Complex 5
N3-H ...O2
0.82(3)
2.646(2)
1.93(3)
144(3)
15

In complex 5 (Fig. 3) the Cu atom is bound to two niflumate and two  -picoline ligands; the
niflumate acts as a bidentate ligand coordinated via the carboxylate group, with very different
Cu-O distances (Table 2), one much longer than the other. The resulting geometry is highly
distorted octahedral.
It is worth mentioning that in all the three structures there is the formation of a quite strong
charge-assisted intramolecular N-H…O hydrogen bond (Table 2) involving the NH and
carboxylate groups of the niflumate moiety, the only difference consisting in the fact that in 1
this intramolecular interaction involves the non-coordinated niflumate oxygen atom.
Fig. 1. ORTEPIII view and atom numbering scheme for complex 1 [Cu(en)2(nif)2].2H2O.
Thermal ellipsoids are drawn at the 40% probability level. Hydrogen bonds are drawn as dashed
lines.
16

Fig. 2. ORTEPIII view and atom numbering scheme for complex 3: [Cu(pn)2(nif)2].
[Cu(pn)2(H2O)2](nif)2 showing the two different Cu coordinations (arbitrary projections).
Thermal ellipsoids are drawn at the 40% probability level. Hydrogen bonds are drawn as dashed
lines. Intramolecular hydrogen bonds are not shown for clarity.
Fig. 3. ORTEPIII view and atom numbering scheme for complex 5 [Cu(-picoline)2(nif)2].
Thermal ellipsoids are drawn at the 40% probability level. Hydrogen bonds are drawn as dashed
lines. Only one position of the disordered CF3 group is shown for clarity.
17

Since it was not possible to obtain a good refined structure of the [Cu(-picoline)2(nif)2]
complex 4, its geometry has been obtained via DFT calculations and it is shown in Fig. 4. Here,
some relevant bond distances are also reported. The geometry is very similar to that of the
related complex 5, including the presence of the strong intramolecular N-H…O hydrogen bond
(N…O distance= 2.658 Å).
Fig. 4. DFT optimized geometry of complex 4 [Cu(-picoline)2(nif)2]. Hydrogen bonds are
drawn as broken lines; Cu-N in blue.
In 1, due to the abundance of good donors and acceptors, the packing diagram is mainly
determined by the formation of O/N-H…O hydrogen bonds. The projection of the unit cell along
a (Fig. 5) clearly shows the formation of a Cu polyhedra/organic layered structure; the
cocrystallized water molecules have the task of joining adjacent complexes, acting both as
hydrogen bond donors towards the oxygens of the carboxylate group, with the formation of a
R4,4(12) ring (Fig.5b), and as acceptors for the NH2 groups. In 3, the hydrogen bonding network
is even more complicated. In addition to the long list of intermolecular interactions reported in
Table 2, a short contact O1W...O3( x,1/2-y,z-1/2) of 2.682(8) Å is indicative of an hydrogen
bond involving the coordinated water molecule. Overall, the packing diagram is made of rows of
18

alternating Cu1/Cu2 complexes as shown in Fig. 6; two adjacent Cu1/Cu2 different complexes
are linked to each other through N-H…O hydrogen bonding interactions involving O2 as
acceptor and N5 and N6 as donors (Table 2).
As for complex 5, no intermolecular hydrogen bond of some importance was observed, due to
the lack of good donor and acceptor groups.
b
a
Fig. 5. (a) Unit cell content of 1 (viewed along the a axis); (b) R4,4(12) ring. Hydrogen bonds
are drawn as broken lines.
Fig. 6. Unit cell content of 3. Polyhedra around the Cu1 and Cu2 atoms are shown in blue and
magenta, respectively.
19

3.3 FT-IR and electronic spectroscopy
Infrared spectra of complexes 1-5 were recorded in the region 4000-400 cm-1 and tentative band
assignments have been made on the basis of earlier reports in the literature [46, 47]. In the solid
state FT-IR spectra of complexes 1-5, significant broad peaks are observed in the region 3500-
31000 cm-1, indicating the O-H and N-H stretching frequency of water and niflumate or nitrogen
donor ligands (such as en, pn, usym-dmen) in these complexes. The absorption bands observed in
the region 3100-2900 cm-1 were assigned to C(sp2)-H stretching vibrations of the niflumate anion
in all complexes. The absorption peaks in the region 1650-1600 cm-1 were assigned to C=C
stretching vibrations of the niflumate molecule. The sharp bands in the region 1600-1500 cm-1
and 1400-1340 cm-1 correspond to the νas(COO) and νs(COO) stretching vibrations of the
carboxylate group of niflumate [8, 13, 46, 47]. The parameter Δν (νas(COO)-νs(COO)) can be used as
an important tool in assigning the mode of coordination of the carboxylate ligand in metal-
carboxylate complexes. From various coordination modes of carboxylate coordination, i) ionic,
ii) unidentate, iii) bidentate chelating are the most common. In complexes 1-3, the Δν(COO) values
of 207, 194, 175 cm-1 , respectively, fall in the range (210-160 cm-1) observed for various ionic
or monodentate complexes e.g. sodium formate (Δν=201cm-1), sodium acetate (Δν=164 cm-1),
sodium ibuprofenate (Δν=190 cm-1), sodium diclofenate (Δν=170) [48]. etc. For complexes 4
and 5, the higher value of Δν = 251, 222 cm-1 , respectively, indicates a bidentate chelation
mode of the carboxylate anion representing covalent nature of these complexes. The absorption
peaks observed in the region 1000-620 cm-1 might be assigned to in-plane bending and out-of-
plane deformation vibrations of hydrogen atoms on aromatic rings. The absorption peaks
observed around 500 cm-1 in complexes 1-5 are within the range reported for Cu-O and Cu-N
20

stretching frequencies in the literature. The FT-infrared spectra for complexes 1-5 are shown in
Fig.S1 (supplementary data).
The UV-Visible spectra of complexes 1-5 were recorded using methanol-water (4:1) as solvent
and exhibit very broad d-d bands in the visible region from 500-800 nm, corresponding to an
octahedral complex or a combined merged broad peak corresponding to a square pyramidal or
square planar geometry. So, the exact geometry of the complex could not be predicted from
solution state UV-Vis spectra. UV-Vis spectra of complexes 1-5 showed a broad absorption band
at 575 nm (ε= 58 L.mol-1.cm-1), 560 (ε= 162 L.mol-1.cm-1), 563 (ε= 188 L.mol-1.cm-1), 728 (ε=
108 L.mol-1.cm-1) nm 735 (ε= 137 L.mol-1.cm-1) nm for complexes 1-5, respectively, indicating
that complexes 1-3 have a similar chromophore and complexes 4 and 5 have different
chromophore. The observed UV-vis of complex 3 shows only one broad absorption band in the
region 570-550 nm indicating the presence of a CuN4O2 chromophore, so it is not possible to
distinguish the two types of coordination environments around the two copper(II) atoms in
asymmetric unit as revealed by the X-ray structure determination. The UV-visible spectra of all
complexes 1-5 are shown in Fig. S2 (supplementary data). The observed electronic spectral
bands for complexes 1-5 are in good agreement with literature data [49]. To investigate charge
transfer transitions, UV-visible spectra of micromolar solutions (water-methanol as solvent, 4:1
v/v) of all complexes were recorded in the range 400-200 nm. The observed peaks at around 290
nm for all complexes correspond to a charge transfer transition between the  electron cloud of
the niflumate moiety and the copper(II) metal ion as shown in Fig. S3 (supplementary data). In
order to investigate variations in coordination in solution with respect to the solid state, UV-vis
spectra in the solid state of complexes 1-5 have been recorded: they do not show any significant
change in absorption maxima in comparison to the solution state, indicating an identical
21

coordination geometry of the copper(II) complexes in solution state. This is in accordance with a
relative stability of the complexes in solution, since no modification in the UV-visible spectra
was observed over time. The solid state UV-Vis spectra of complexes 1-5 are shown in Fig.S3
(supplementary data).
3.4 EPR Spectroscopy
The EPR spectra of Cu(II) complexes 1-5, together with the reference complex Cu(nif)2.2H2O,
obtained in CHCl3 solution at 110 K at X-band (9.43 GHz) demonstrate a typical pattern of
copper complexes with hyperfine structure resulting from the coupling of the Cu2+ electron spin
(S=1/2) with its nuclear spin (I=3/2) (Fig. S5 of the supplementary data). The obtained spin-
Hamiltonian parameters (g//, A// and A┘, g┘), reported in Table 3, are typical for an axially
2
elongated dx2-y ground state (g|| > g┴ ≈ 2.0023) for Cu2+ ions with an octahedral configuration
geometry around the central ion. Two complexes “families” can be evidenced according to these
parameters. Indeed, Kivelson and Neiman [50] have reported that g|| values less than 2.3 indicate
considerable covalent character of the metal-ligand bonds, while a value greater than 2.3
indicates an ionic character. As the g|| value of complexes 1, 2, 3, and Cu(nif)2.2H2O is found to
be less than 2.3 it implies a significant covalent character of the metal-ligand bonds in these
complexes. This is in line with the monodentate binding mode of the niflumate ligand observed
in these cases. On the other hand, in view of the Peisach-Blumberg diagram [51], their obtained
values of g// and A// are consistent with a CuN4O2 chromophore (except for Cu(nif)2.2H2O). In
contrast, in the case of complexes 4 and 5, including a picoline ligand and where the niflumate
ligand is bidentally coordinated (X-ray crystallography section), the g|| value higher than 2.3
indicates an ionic character of the Cu2+-niflumate bonds. Moreover, following the Peisach-
22

Blumberg diagram, their spin-Hamiltonian parameters underline a CuO4N2 chromophore.
Finally, the EPR parameters of Cu(nif)2.2H2O are typical of a CuO4 chromophore.
Table 3. EPR parameters for copper complexes 1-5 and the Cu(nif)2.2H2O reference complex
Complex
Tensor
10-4 cm-1
10-4 cm-1
Cu(nif)2.2H2O
g┘ = 2.085
g// = 2.271
A┘ = 23
A// = 181
LW┘ = 32
LW// = 42
Complex 1
Complex 2
Complex 3
Complex 4
g┘ = 2.032
g// = 2.229
A┘ = 16
A// = 196
LW┘ = 25
LW// = 29
g┘ = 2.045
g// = 2.242
A┘ = 18
A// = 191
LW┘ = 28
LW// = 45
g┘ = 2.035
g// = 2.251
A┘ = 19
A// = 187
LW┘ = 30
LW// = 38
g┘ = 2.040
g// =2.321
A┘ =25
A// = 147
LW┘ = 47
LW// = 39
Complex 5
g┘ = 2.042
g// =2.326
A┘ =26
A// = 149
LW┘ = 51
LW// = 40
3.5 Biological activity
In vitro assays using mammalian cell cultures provide valuable information about the toxicity of
compounds and can be used as an accurate approach to estimate acute toxicity. The basal
cytotoxic effects of the copper complexes were tested by a colorimetric assay on 2 human
melanoma cell lines HT-144 and SKMel -28, which is a first step for further toxicity studies. Cell
viability and IC50 values of complexes in HT-144 and SKMel -28 cells are reported in Figure 7
and Table 4. All Cu-niflumate complexes showed higher cytotoxicity than the parent molecule
(IC50 > 500 μM, data not shown) against the two human cell cultures. While [Cu2(nif)4.2H2O],
complexes 1 and 2 exhibit a similar activity towards the two tumoral cell lines with low
23

cytotoxicity (IC50 higher than 120 µM), comparison of the cytotoxic activities of complexes 3-5
indicates a differential cytoxycity toward the two cell lines. Each complex shows a significant
cytotoxicity on the human melanoma cell line HT-144 (with a mean IC50 of 69 µM for complex
3, 50 µM for complex 4 and 33 µM for complex 5) whereas weak cytotoxic activities of the
complexes are observed on the human melanoma cell line SKMel-28 with a mean IC50 higher
than 130 µM for the 3 complexes. Previous work demonstrated that similar Cu(II)-Ibuprofen
complexes induce significant cytotoxicity on the HT-144 cell line whereas a weak cytotoxic
activity of the complexes is observed on the healthy human dermal fibroblast HDF cells, and the
human melanoma cell line SKMel-28, too [33]. It is interesting to note that in the case of
complexes 4 and 5, showing the highest cytotoxicity, the niflumate ligand is bidentate whereas in
the case of the other complexes it is monodentate; moreover, as mentioned above, in complex 1,
at variance with the other cases, the intramolecular NH…O hydrogen bond involves the non-
coordinated niflumate oxygen. These structural characteristics could explain the cytotoxicity
differences.
24

Table 4. IC50 of complexes in HT-144 and SKMel-28 cells at 24h as determine by a colorimetric
assay. Data are expressed as IC50 values (µM) and are means ± SD of 3 independent experiments.
IC50 (µM)
Complexes
HT-144
SKMel-28
Cu(nif)2.2H2O (S0)
Complex 1 (S1)
Complex 2 (S2)
Complex 3 (S3)
Complex 4 (S4)
Complex 5 (S5)
141 ± 8
124 ± 13
159 ± 6
69 ± 9
158 ± 7
162 ± 8
163 ± 7
147 ± 9
137 ± 9
134 ± 9
> 200
50 ± 8
34 ± 8
113 ± 23
Cu(Ibu)2.xH2O [33b]
25

SkMel
28
200
150
100
50
S0
S1
S2
S3
S4
S5
0
0
6.25
12.5
25
50
100
200
Concentration (µM)
HT14
120
100
80
60
40
20
0
4
S0
S1
S2
S3
S4
S5
0
6.25
12.5
25
50
100
200
Concentration (µM)
Fig. 7. Cytotoxic effects of the complexes on tumoral (human dermal melanoma cell lines HT-
144 and SKMEI-28) human cell lines. Each cell line was treated (0-200 µM) with Cu(II)
complexes for 24h and viable cells were determined using a colorimetric assay. Each
concentration point represents the mean ± S.E.M. of triplicate samples.
26

4. Conclusions
Five new copper(II)-niflumate complexes 1-5 have been synthesized in the presence of
different nitrogen-donor ligands using methanol-water (4:1 v/v) as solvent in nearly quantitative
yield at room temperature. The newly synthesized copper(II) complexes were characterized by
elemental analyses and spectroscopic methods (UV-Vis, FT-IR, EPR). The structures of
complexes 1, 3 and 5 have been unambiguously determined by X-ray structure determination,
confirming the formation of mononuclear covalent complexes. The structure of complex 4 has
been optimized with the help of DFT calculations which provided insight into the possible
existence of a structure similar to that of complex 5. The EPR spectra revealed a typical pattern
of copper complexes with hyperfine structure resulting from the coupling of the Cu2+ electron
spin (S=1/2) with its nuclear spin (I=3/2). The g|| < 2.3 for complexes 1-3 imply covalent
character which is in line with the monodentate coordination mode of the anionic niflumate
ligand, whereas the values of g// and A// for complex 4 and 5 is consistent with a CuN4O2
chromophore. Complexes in which the niflumate ligand is bidentate (4 and 5) showed
significantly higher cytotoxicity against the human melanoma cell line HT-144 than the other
complexes (1-3), where niflumate exhibits a monodentate coordination mode.
Acknowledgements
The authors RPS and SK acknowledge the financial support from UGC, New Delhi
(India) as a UGC Emeritus and BSR Meritorious Fellowship, respectively. PV thanks the DST
PURSE program of Panjab University, Chandigarh
Supplementary Data
Supplementary data content: details of the IR, UV-Vis and EPR spectra (5 figures) and X-ray
crystallography (1 table).
27

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