Synthesis, crystal structures and spectroscopy of meclofenamic acid and its metal complexes with manganese(II), copper(II), zinc(II) and cadmium(II). Antiproliferative and superoxide dismutase activity

Article abstract of DOI:10.1016/j.jinorgbio.2011.05.025

Some new complexes of meclofenamic acid (N-(2,6-dichloro-m-tolyl) anthranilic acid), Hmeclo (1), with potentially interesting biological activities are described. Complexes [Mn(meclo)₂] (2), [Cu(meclo) ₂(H₂O)₂] (3), [Zn(meclo)₂(H ₂O)₂] (4) and [Cd(meclo)₂(H₂O) ₂] (5) were prepared and structurally characterized by means of vibrational, electronic and ¹H and ¹³C NMR spectroscopies. The crystal structure of complexes [Cu₄(meclo)₆(OH) ₂(DMSO)₂]2DMSO (3a) and [Cd(meclo)₂(DMSO) ₃] (5a) have been determined by X-ray crystallography. Complex (3a) is a centrosymmetric tetramer built up around the planar cyclic Cu ₂(OH)₂ unit. Complex 5a is mononuclear seven-coordinated complex with the meclofenamato ligand behaving as a bidentate deprotonated chelating ligand. Intra and intermolecular hydrogen bonds stabilize these two structures, while the crystal packing is determined by π-π and C-H - π interactions. Meclofenamic acid and its metal complexes have been evaluated for antiproliferative activity in vitro against the cells of three human cancer cell lines, MCF-7 (breast cancer cell line), T24 (bladder cancer cell line), and A-549 (non-small cell lung carcinoma), and a mouse fibroblast L-929 cell line. Complex 5 exhibits the highest selectivity against MCF-7 and 4 shows the highest selectivity against T-24. Complexes 2-5 were found to be more potent cytotoxic agents against T-24 and complex 5 against MCF-7 cancer cell lines than the prevalent benchmark metallodrug, cis-platin. The superoxide dismutase activity was measured by the Fridovich test which showed that complex [Cu(meclo) ₂(H₂O)₂] is a good superoxide scavenger.

Full text of DOI:10.1016/j.jinorgbio.2011.05.025

Journal of Inorganic Biochemistry 105 (2011) 1187–1195
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, crystal structures and spectroscopy of meclofenamic acid and its metal
complexes with manganese(II), copper(II), zinc(II) and cadmium(II).
Antiproliferative and superoxide dismutase activity
a
b
Dimitra Kovala-Demertzi a, , Malgorzata Staninska , Isabel Garcia-Santos ,
⁎
Alfonso Castineiras b, Mavroudis A. Demertzis a
a
Inorganic and Analytical Chemistry, Department of Chemistry, University of Ioannina, 45100 Ioannina, Greece
Departamento de Quımica Inorganica, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Some new complexes of meclofenamic acid (N-(2,6-dichloro-m-tolyl)anthranilic acid), Hmeclo (1), with
potentially interesting biological activities are described. Complexes [Mn(meclo)2] (2), [Cu(meclo)2(H2O)2]
(3), [Zn(meclo)2(H2O)2] (4) and [Cd(meclo)2(H2O)2] (5) were prepared and structurally characterized
by means of vibrational, electronic and 1H and 13C NMR spectroscopies. The crystal structure of complexes
[Cu4(meclo)6(OH)2(DMSO)2]•2DMSO (3a) and [Cd(meclo)2(DMSO)3] (5a) have been determined by X-ray
crystallography. Complex (3a) is a centrosymmetric tetramer built up around the planar cyclic Cu2(OH)2 unit.
Complex 5a is mononuclear seven-coordinated complex with the meclofenamato ligand behaving as a
bidentate deprotonated chelating ligand. Intra and intermolecular hydrogen bonds stabilize these two
structures, while the crystal packing is determined by π–π and C−H−–π interactions. Meclofenamic acid and
its metal complexes have been evaluated for antiproliferative activity in vitro against the cells of three human
cancer cell lines, MCF-7 (breast cancer cell line), T24 (bladder cancer cell line), and A-549 (non-small cell lung
carcinoma), and a mouse fibroblast L-929 cell line. Complex 5 exhibits the highest selectivity against MCF-7
and 4 shows the highest selectivity against T-24. Complexes 2–5 were found to be more potent cytotoxic
agents against T-24 and complex 5 against MCF-7 cancer cell lines than the prevalent benchmark metallodrug,
cis-platin. The superoxide dismutase activity was measured by the Fridovich test which showed that complex
[Cu(meclo)2(H2O)2] is a good superoxide scavenger.
Received 1 March 2011
Received in revised form 30 May 2011
Accepted 31 May 2011
Available online xxxx
Keywords:
Metal complexes
Meclofenamic acid
Crystal structures
Spectroscopic studies
Antineoplastic activity
Sod activity
© 20112011 Elsevier Inc. All rights reserved.
1. Introduction
channels through a pathway that may be independent of the
cyclooxygenase–prostaglandin mechanism [2]. It was found that
Meclofenamic acid (N-(2,6-dichloro-m-tolyl)anthranilic acid),
HMeclo, is a non-steroidal anti-inflammatory drug (NSAID) from the
family of fenamates with marked analgesic properties and is used in the
treatment of osteoarthritis (OA), rheumatoid arthritis (RA) and other
painful musculoskeletal diseases. Chemically, it resembles mefenamic
and flufenamic acids and other fenamates in clinical use. The mode of
action of the NSAIDs is attributed primarily to the inhibition of
prostaglandins (PG) synthesis, and more specifically inhibition of the
cyclo-oxygenase enzyme system, Cox, Cox-1 and Cox-2. Inhibition of the
Cox-2 system results in anti-inflammatory action, while inhibitionof the
Cox-1 enzyme system results in anti-inflammatory action as well as
gastric irritation [1]. However, accumulating evidence indicates that
fenamates and meclofenamic acid also modulate a diversity of ion
meclofenamic acid results in the generation of potent and selective
Cox-2 inhibitors [1]. New studies from the last years revealed that in
addition to arthritis and pain, cancer and neurodegenerative diseases
like Alzheimer's disease could potentially be treated with Cox-2
inhibitors [3,4]. Some researchers propose that NSAIDs, in addition to
their inhibitory effects on the synthesis of PGs, may also inhibit the
production, or act as scavengers of free radicals [5–8].
Metal complexes with active drugs as ligands are a research area of
increasing interest for inorganic and medicinal chemistry and have
concentrated much attention as an approach to new drug development
[9,10]. The synthesis of metal complexes with NSAIDs as ligands has
acquired new impetus in the past decade. The information collected
from the preparative, structural and reactivity studies have high
significance for several fields which span from the bio-sciences to the
material sciences. The combination of two or more different species into
the same compound may bring to a multi-therapeutic agent which can
be expanded by the synergic action of the metal residue once the co-
ordination compound dissociates inside the target tissue [12,13,15,17].
⁎
Corresponding author. Tel.: +30 2651008425; fax: +30 2651008791.
E-mail address: dkovala@cc.uoi.gr (D. Kovala-Demertzi).
0162-0134/$ – see front matter © 20112011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.05.025

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D. Kovala-Demertzi et al. / Journal of Inorganic Biochemistry 105 (2011) 1187–1195
The coordination chemistry of NSAIDs has been studied by several
groups worldwide. Some complexes have increased pharmaceutical or
biological activity with respect to the drug or are interesting from a
purely chemical point of view [10–17]. A review of the synthesis and
crystal structures of a number of anti-inflammatory compounds as
ligands in organotin complexes and a review of copper complexes with
NSAIDs have been reported [6,10,14,16]. Crystal structures of organotin
meclofenamic complexes were reported by our group. The [Ph3Sn
(Meclo)] complex exhibited high cytotoxic activity against cancer cell
lines and was found to be a promising anti-mycobacterial lead
compound, displaying high activity against M. tuberculosis H37Rv [17].
Copper(II)-complexes, including Cu-NSAIDs, exhibit significant
anti-inflammatory activities as well as superoxide dismutase (SOD)
mimetic activity. SODs, a family of metalloenzymes, catalyses the
dismutation of superoxide radical anions to the non-radical products
oxygen and hydrogen peroxide and protects living cells from damage
induced by reactive oxygen species (ROS). The use of SOD as a
pharmaceutical has been proposed for treatment of a number of
diseases including, hyperoxia, reperfusion injury, acquired immune
deficiency syndrome (AIDS) disease auto-immune deficiency disease
(AIDS), ulcerative colitis, bronchopulmonary dysplasia in premature
neonates, as well as inflammation and inflammation-associated
diseases, such as rheumatoid arthritis (RA) and osteoarthritis (OA)
[6]. Although there is much interest in the use of SOD for pharmaceutical
purposes, its practical use in biological systems is problematic due to
difficulties associated with the systematic infection of protein, i.e. the
circulation lifetime, cell impermeability, immunogenicity, tissue target-
ing, antigenicity and highcosts. To avoid such limitations, there has been
considerable interest in developing synthetic SOD mimics that have low
molecular weight, biological stability, and membrane permeability and
are nontoxic and cost-effective [6,18,19]. However, the development of
new synthetic compounds that act as mimetics of SOD and catalase has
offered an alternative approach with some promise. Use of such
compounds in mouse models has been effective in attenuating oxidative
stress-associated disease processes and leads to extension of lifespan
[20,21].
We have prepared novel complexes of meclofenamic acid,
Scheme 1, with Mn(II), Cu(II), Zn(II) and Cd(II), [Mn(meclo)2],
[Cu(meclo)2(H2O)2], [Zn(meclo)2(H2O)2], and [Cd(meclo)2(H2O)2]
in order to obtain information on structure–activity relationships for
systems involving metal atoms. The cytotoxic activity of meclofenamic
acid and its metal complexes have been evaluated for antiproliferative
activity in vitro against the cells of three human cancer cell lines: MCF-7
(human breast cancer cell line), T24 (bladder cancer cell line), A-549
(non-small cell lung carcinoma) and a mouse L-929 (a fibroblast-like
cell line cloned from strain L). Also, the superoxide dismutase activity
was measured and IC50 value was determined by the Fridovich test [22].
Herein, the goal is to define the probability to extend the pharmaco-
logical profile of meclofenamic acid, in order to discover new properties
such as anti-cancer activity and SOD activity, to prepare new
compounds, complexes of meclofenamic acid with essential metal
ions, which probably would exhibit improved or different biological
and pharmaceutical behaviour compared to the “parent drug”,
meclofenamic acid.
2. Experimental section
2.1. General and instrumental
All reagents were commercially available (Aldrich or Merck) and
used as supplied. Melting points were determined in open capillaries and
are uncorrected. Infrared and far-infrared spectra were recorded on a
Perkin Elmer Spectrum GX Fourier transform spectrophotometer using
KBr pellets (4000–400 cm−1) and nujol mulls dispersed between
polyethylene disks (400–40 cm−1). ΝΜR spectra were recorded on a
Bruker AV-400 spectrometer operating at 400 and 100 MHz for 1H and
13C acquisition, respectively, or on a Bruker AV-250 spectrometer
operating at 250.13 and 62.90 MHz for 1H and 13C acquisition
respectively. The spectra were acquired at room temperature (298 K).
The splitting of proton resonances in the reported 1H NMR spectra are
defined as s=singlet, d=doublet, t=triplet, and m=multiplet. The
chemical shifts are reported in ppm for 1H and 13C NMR. Samples were
dissolved in CDCl3 or DMSO-d6 and spectra were obtained at room
temperature with the signal of the free DMSO or CHCl3 (at 2.49 and
7.24 ppm, respectively) as a reference. Elemental analyses, C, H, N and S
were performed on a Carlo Erba EA (model 1108). Mass spectra were
measured on an Agilent 1100 Series LC-MSD-Trap-SL spectrometer.
Experimental and calcd. MS (ESI-MS) values (m/z) for each of the five
compounds were identical to a significant last figure above the decimal
point. Metal compositions were determined using an energy-dispersive
X-ray fluorescence (EDXRF) spectroscopy arrangement.
2.2. Preparation of HMeclo and the complexes
2.2.1. Synthesis 2-[(2,6-Dichloro-3-methyl-phenyl)amino]benzoic acid
(HMeclo); (1)
Meclofenamic was synthesized according to a published pro-
cedure from (2-carboxyphenyl)phenyliodonium and 2,6-dichloro-
3-methylbenzenamine [23]. The white power was collected and
recrystallized three times from ethanol to afford 1, Yield 70%. M.p.
252–253 °C. IR (KBr): 3336 (ν(ΝΗ)); 3060br (ν(OΗ)); 2920, 2860
(ν(CΗ3)); 1656 (νasym(COO)); 1438(νsym(COO)); UV–visible (UV–
vis): λ, nm (ε/logε) in DMF: 319 (3.66), 291 (3.64); in CHCl3: 340
(3.53), 282 (3.49); 1H NMR (DMSO): 13.14 s (OH); 9.15 (s, NH);
7.90 (dd, H–C(3), JH(3)–H(4) =8.0, JH(3)–H(5) =1.0); 6.77 (t, H–C(4),
JH(4)–H(5) =8.0, JH(4)–H(6) =1.1); 7.31 (m, H–C(5)), 6.19 (d, H–C(6),
JH(6)–H(5) =8.4); 7.35 (d, H4′), JH(4′)–H(5′) =8.3); 7.50 (d, H5′),
JH(5′)–H(4′) = 8.3); 2.38 (s, 3′ Me); 13C NMR: 170.0 (COOH); 147.1
(C(1)); 111.7 (C(2)); 131.5 (C(3)); 117.3 (C(4)); 134.6 (C(5));
112.9 (C(6)); 134.1 (C(1′)); 133.5 (C(2′)); 130.5 (C(3′)); 129.3 (C(4′));
128.8 (C(5′)); 130.6 (C(6′)); 20.1 (3′ Me); 1H NMR (CDCl3): 9.21
(s, NH); 8.07 (d, H–C(3), JH(3)–H(4)=6.7); 6.79 (t, H–C(4), JH(4)–H(5)
=
7.6, JH(4)–H(6)=1.1); 7.22 (m, H–C(5)), 6.33 (d, H–C(6), JH(6)–H(5) =8.4);
7.14 (d, H4′), JH(4′)–H(5′) =8.3); 7.32 (d, H5′), JH(5′)–H(4′) =8.2);
2.41 (s, 3′ Me); 13C NMR: 172.1 (COOH); 148.5 (C(1)); 110.4
(C(2)); 132.3 (C(3)); 117.4 (C(4)); 134.9 (C(5)); 113.1 (C(6)); 135.1
(C(1′)); 134.7 (C(2′)); 131.6 (C(3′)); 128.8 (C(4′)); 127.8 (C(5′)); 136.6
(C(6′)); 20.6 (3′ Me); Mass spectrum, MS (electrospray ionization,
ESI, m/z): 297 [1+H]+, 612[12 +H3O]+. Anal. calc. for C14H11Cl2NO2
(296.13 g mol–1): C, 56.8; H, 3.7; N, 4.7; found: C, 56.2, H, 3.9; N, 4.3%.
2.2.2. Synthesis of [Mn(meclo)2]; (2)
Anhydrous manganese(II) acetate ([Mn(CH3COO)2]) (0.0216 g,
0.125 mM) was dissolved in methanol (2 mL) and this solution was
added to a solution of meclofenamic acid (0.0740 g, 0.25 mM) in
methanol (2 mL). Drops of triethylamine (N(eth)3) were added to the
solution till the apparent pH value was ~7. The reaction mixture was
stirred for 4 h at room temperature and then it was left to the
refrigerator overnight. Drops of cold distilled water were added to the
solution and a whitish precipitate was collected by filtration. The
powder was filtered and washed with cold MeOH/H2O 1/1 (1:1), and
Scheme 1. The numbering scheme for Hmeclo, 1.

D. Kovala-Demertzi et al. / Journal of Inorganic Biochemistry 105 (2011) 1187–1195
1189
dried in vacuo to afford (2), yield 47%. M.p. 150–151 °C IR (cm−1):
3266 m, ν(NH); 3070 m 2983 m, ν(CH3); 1615 s, νasym(COO); 1394 s,
yield 95%. M.p. 191–192 °C. IR (cm−1): 3251 m, ν(NH); 3068 m 2986 m,
ν(CH3); 1618 s, νasym(COO); 1390 s, νsym(COO); 430 ms, ν(Cd-OH2O);
368 m, 304 m, ν(Cd-Ooco). 1H NMR (DMSO): 10.33 (s, NH); 8.02 (dd,
H–C(3), JH(3)–H(4) =7.4); 6.74 (t, H–C(4), JH(4)–H(5) =7.4); 7.21 (t,
H–C(5)), JH(5)–H(45) =7.4);), 6.15 (d, H–C(6), JH(6)–H(5) =8.1); 7.32
(d, H4′), JH(4′)–H(5′) =8.2); 7.48 (d, H5′), JH(5′)–H(4′) =8.2); 2.38 (s,
3′ Me); 13C NMR: 173.9 (COOH); 146.1(C(1)); 131.9 (C(3)); 116.7
(C(4)); 135.5 (C(5)); 112.2 (C(6)); 133.3 (C(1′)); 132.4 (C(2′));
130.2 (C(3′)); 128.6 (C(4′)); 128.0 (C(5′)); 136.3 (C(6′)); 20.2 (3′ Me);
νsym(COO); 438 ms, ν(Mn-OH2O); 366, 277 ms, ν(Mn-Ooco). UV–vis:
λ, nm (ε/logε) in DMF:415sh (1.51), 364 (2.37), 321 (4.10), 294
(4.08); in CHCl3: 493sh (1.63), 381 (2.46), 337 (3.84), 283 (3.85).
μ
eff =5.66 MB. Mass spectrum, MS (atmospheric pressure chemical
ionization, APCI, m/z): 664 [2+H3O]+, 296 [1+H]+. Anal. calc. for
C28H20Cl4MnN2O4 (645.2 g mol–1): C, 52.1; H, 3.1; N, 4.3; Mn, 8.5
found: C, 52.3, H, 3.2; N, 4.2; Mn, 8.8%. The complex is soluble in
DMSO, DMF, MeOH, Benzene, toluene, CH2Cl2, and CHCl3.
Mass spectrum, MS (electrospray ionization, ESI, m/z): 632 [5-2Cl− +
] ,
764 [5+Na]+, 741[5+2H]+. Anal. calc. for C28H24CdCl4N2O6
(738.67.6 g mol–1): C, 45.5; H, 3.3; N, 3.8; Zn, 15.2 found: C, 45.6, H,
3.4; N, 3.9; Zn, 15.3%. The complex is soluble in DMSO and DMF and
slightly soluble in CH2Cl2, CHCl3. Recrystallization of 5 from DMSO
solution gives single crystals of [Cd(meclo)2(DMSO)3] (5a) suitable for
X-ray structure determination.
2.2.3. Synthesis of [Cu(meclo)2(H2O)2]; (3)
Copper(II) acetate monohydrate ([Cu(CH3COO)2(H2O)]2)
(0.0250 g, 0.125 mM) was dissolved in methanol (2 mL) and this
solution was added to a solution of meclofenamic acid (0.0740 g,
0.25 mM) in methanol (2 mL). Drops of N(eth)3 were added to the
solution till the apparent pH value was ~7. The reaction mixture was
stirred for 4 h at room temperature and then it was left to the
refrigerator overnight. The green powder was filtered and washed
with cold MeOH and dried in vacuo to afford (3), yield 69%. D.p. 190–
192 °C. IR (cm−1): 3312 m, ν(NH); 3070 m 2923 m, ν(CH3); 1618 s,
2.3. X-ray crystallography
A green needle crystal of [Cu4(meclo)6(OH)2(DMSO)2]•2DMSO (3a)
and a colourless plate crystal of [Cd(meclo)2(DMSO)3] (5a) were
mounted on a glass fiber and used for data collection. Crystal data were
collected at 100(1) K, using a Bruker X8 KappaAPEXII diffractometer and
a Bruker SMART CCD 1000 diffractometer for 3a and 5a respectively.
Graphite monochromated MoK(alpha) radiation was used throughout.
The data were processed with APEX2 and with SAINT for 3a and for 5a
respectively. The data were corrected for absorption using SADABS
(transmissions factors: 1.000–0.0.904) and (transmissions factors:
1.000–0.887) for 3a and for 5a respectively [24]. The structures were
solved by direct methods using the program SHELXS-97 and refined by
full-matrix least-squares techniques against F2 using SHELXL-97 [25].
Positional and anisotropic atomic displacement parameters were
refined for nonhydrogen atoms. Hydrogen atoms were located in
difference maps and included as fixed contributions riding on attached
atoms with isotropic thermal parameters 1.2 times those of their carrier
atoms. The H atoms of methyl groups and isotropically refined atoms
were included in geometrically idealized positions. Criteria of a
satisfactory complete analysis were the ratios of “rms” shift to standard
deviation less than 0.001 and no significant features in final difference
maps. The C atoms of the methyl groups in each ligand for 3a showed
disorder and was refined using a split with 50% occupancy for each
“meta” position (C24/C25, C44/C45 and C64/C65). The lowest
(−1.68 e Å−3) and highest (2.44 e Å−3) peaks for 5a in the final
difference Fourier map are located close to the Cl23 atom at distances of
0.76 and 0.79 Å, respectively. Molecular graphics were performed from
PLATON [26]. A summary of the crystal data, experimental details and
refinement results is listed in Table 1.
νasym(COO); 1391 s, νsym(COO); 480 ms, ν(Cu-OH2O); 375 m, 327 m,
245 ms, ν(Cu-Ooco). UV–vis: λ, nm (ε/logε) in DMF: 733 (2.25), 321
(4.13), 281 (4.16); in CHCl3: 682 (1.83), 339 (4.07), 381 (4.06), 283
(3.85). μeff =1.94 MB.Mass spectrum, MS (Electrospray Ionization,
ESI, m/z): 652 [3-2H3O]−, 295 [1-H]−. Anal. calc. for C28H24Cl4CuN2O6
(689.8 g mol–1): C, 48.9; H, 3.5; N, 4.1; Cu, 9.2 found: C, 49.1, H, 3.2; N,
4.2; Cu, 8.9%. The complex is soluble in DMSO, DMF, MeOH, EtOH,
MeCN, benzene, and toluene and slightly soluble in CH2Cl2 and CHCl3.
Recrystallization of 3 from DMSO solution gives green single-crystals
of the tetramer complex [Cu4(meclo)6(OH)2(DMSO)2]•2DMSO (3a)
suitable for X-ray structure determination.
2.2.4. Synthesis of [Zn(meclo)2(H2O)2]; (4)
Zinc(II) acetate dihydrate ([Zn(CH3COO)2(H2O)2]) (0.0274 g,
0.125 mM) was dissolved in methanol (2 mL) and this solution was
added to a solution of meclofenamic acid (0.0740 g, 0.25 mM) in
methanol (2 mL). The same procedure as in Section 2.2.2 was
followed. Yellow powder (4), yield 29%. M.p. 140–141 °C. IR
(cm−1): 3295 m, ν(NH); 3068 m 2986 m, ν(CH3); 1618 s, νasym
(COO); 1397 s, νsym(COO); 467 ms, ν(Zn-OH2O); 380 m, 303 m,
270 ms, ν(Zn-Ooco). 1H NMR (DMSO): 10.23 (s, NH); 8.00 (d, H–C(3),
JH(3)–H(4)=9.0); 6.67 (t, H–C(4), JH(4)–H(5)=7.4,); 7.16 (m, H–C(5)),
6.12 (d, H–C(6), JH(6)–H(5)=8.2); 7.19 (d, H4′), JH(4′)–H(5′) =8.3);
7.38 (d, H5′), JH(5′)–H(4′)=8.3); 2.30 (s, 3′ Me); 13C NMR: 173.6
(COOH); 146.2 (C(1)); 132.0 (C(3)); 116.7 (C(4)); 135.5 (C(5)); 112.3
(C(6)); 133.3 (C(1′)); 132.1 (C(2′)); 130.2 (C(3′)); 128.5 (C(4′)); 127.9
(C(5′)); 136.2 (C(6′)); 20.1 (3′ Me); 1H NMR (CDCl3): 9.42 (s, NH);
8.00 (d, H–C(3)); 6.63 (t, H–C(4), JH(4)–H(5) =7.2); 7.17 (d, H–C(5),
J(5–4)=8.1), 6.25 (d, H–C(6), JH(6)–H(5) = 8.3); 6.97 (d, H4′),
2.4. Antiproliferative assay in vitro
JH(4′)–H(5′) = 8.3); 7.21 (d, H5′), JH(5′)–H(4′)=8.2); 2.28 (s, 3′ Me); 13
C
Compounds: Test solutions of the compounds tested (1 mg/mL)
were prepared by dissolving the substance in 100 μL of DMSO
completed with 900 μL of tissue culture medium. Afterwards, the
tested compounds were diluted in culture medium to reach the final
concentrations of 100, 50, 10, 1 and 0.1 ng/μL. The solvent (DMSO) in
the highest concentration used in test did not reveal any cytotoxic
activity.
NMR: 175.2 (COOH); 147.25 (C(1)); 113.6 (C(2)); 131.1 (C(3)); 117.1
(C(4)); 135.9 (C(5)); 115.2 (C(6)); 134.3 (C(1′)); 133.3 (C(2′)); 129.1
(C(3′)); 127.0 (C(4′)); 127.9 (C(5′)); 136.2 (C(6′)); 20.5 (3′ Me); Mass
spectrum, MS (APCI, m/z): 728 [4+2HO]−, 295 [1-H]−. Mass spectrum,
MS (Electrospray Ionization, ESI, m/z): 753 [4+2CH3O− −
]
, 1345 [42-
2H3O]− 569 [4-2Cl–CH3]+
, . Anal. calc. for C28H24Cl4N2O6Zn
(691.6 g mol–1): C, 48.6; H, 3.5; N, 4.1; Zn, 9.5 found: C, 48.4, H, 3.4; N,
4.2; Zn, 9.6%. The complex is soluble in DMSO and DMF and slightly
soluble in CH2Cl2, CHCl3.
2.4.1. Cells
The cell lines are maintained in the Cell Culture Collection of the
University of Ioannina. Twenty-four hours before addition of the
tested agents, the cells were plated in 96-well plates at a density of
104 cells per well. The MCF-7 cells were cultured in the D-MEM
(Modified Eagle's Medium) medium supplemented with 1% antibiotic
and 10% fetal calf serum. L-929 cells were grown in Hepes-buffered
RPMI 1640 medium supplemented with 10% fetal calf serum,
2.2.5. Synthesis of [Cd(meclo)2(H2O)2]; (5)
Cadmium(II) chloride dihydrate (CdCl2•2H2O) (0.1097 g, 0.25 mM))
was dissolved in methanol (2 mL) and this solution was added to a
solution of meclofenamic acid (0.0740 g, 0.25 mM) in methanol (2 mL).
The same procedure as in Section 2.2.3 was followed. Yellow powder (5),

1190
D. Kovala-Demertzi et al. / Journal of Inorganic Biochemistry 105 (2011) 1187–1195
Table 1
Selected crystallographic data for 3a and 5a.
[Cu4(meclo)6(OH)2(DMSO)2] • 2DMSO
92 H80 Cl12 Cu4 N6 O18 S4
[Cd(meclo)2(DMSO)3]
Empirical formula
Formula weight
C
C34 H38 Cd Cl4 N2 O7 S3
937.04
2365.42
Temperature
100(2) K
110(2) K
Wavelength
0.71073
0.71073
Crystal system, space group
Unit cell dimensions
Monoclinic, P 21/n
Triclinic, P −1
a (Å)
8.4916(5)
8.6917(17)
b (Å)
25.2754(17)
9.2754(18)
c (Å)
23.1572(16)
24.612(5)
α (°)
90
85.617(3)
β (°)
93.224(3)
83.067(3)
γ (°)
90
4962.3(6)
2, 1.583
1.322
73.185(3)
1883.7(6)
2, 1.652
1.080
Volume (Å3)
Z, Calculated density (Mg/m3)
Absorption coef. (mm−1
F(000)
)
2404
952
Crystal size (mm)
θ range for data collection (°)
Limiting indices
Reflections collected/unique
Absorption correction
0.47×0.05×0.05
1.19–26.02
−10b=hb=10, 0b=kb=31, 0b=lb=28
74,833/9796 [R(int)=0.0671]
Empirical
0.27×0.23×0.06
0.83–26.37
−10b=hb=10, −11b=kb=11, 0b=lb=30
21,646/7683 [R(int)=0.0348]
Empirical
Max. and min. transmission
Refinement method
0.9369 and 0.5755
Full-matrix least-squares on F2
9796/0/640
0.9380 and 0.7592
Full-matrix least-squares on F2
7683/0/460
Data/restraints/parameter
Goodness-of-fit on F2
1.022
1.091
Final R indices [IN2sigma(I)]
R indices (all data)
R1 =0.0517, wR2 =0.1061
R1 =0.1033, wR2 =0.1300
0.876 and −0.687
R1 =0.0558, wR2 =0.1100
R1 =0.0801, wR2 =0.1218
2.445 and −1.684
Larg. diff. peak and hole (e.Å−3
)
penicillin (50 U/mL) and streptomycin (50 mg/mL). A-549 cells were
grown in F-12K Ham's medium supplemented with 1% glutamine, 1%
antibiotic/antimycotic, 2% NaHCO3 and 10% fetal calf serum. The cell
cultures were maintained at 37 °C in a humid atmosphere saturated
with 5% CO2. Cell number was counted by the Trypan blue dye
exclusion method. MCF-7, L-929 and A-549 cells were determined by
the sulforhodamine B assay [27], while T-24 cells by the MTT assay
[28]. The in vitro tests were performed as described previously [29].
½CuðCH3COOÞ2ðH2OÞꢀ2 þ Hmeclo→½CuðmecloÞ2ðH2OÞ2ꢀ þ 2CH3COOH
ð2Þ
½ZnðCH3COOÞ2ðH2OÞ2ꢀ þ Hmeclo→½ZnðmecloÞ2ðH2OÞ2ꢀ þ 2CH3COOH
ð3Þ
ð4Þ
CdCl2•2H2O þ Hmeclo→½CdðmecloÞ2ðH2OÞ2ꢀ þ 2HCl
Recrystallization of [Cu(meclo)2(H2O)2] (3) from DMSO solution
gives the complex [Cu4(meclo)6(OH)2(DMSO)2]•2DMSO (3a). In the
presence of DMSO a transformation of the monomer (3) to a tetramer
(3a) occurs. In this case hydrolysis of water and aggregation occurs in
the presence of DMSO and that brings about compound 3a according
to the reaction
2.5. Superoxide dismutase assay
SOD activity of complexes 2–5 and the parent drug 1 was
determined by using their ability to inhibit the reduction of nitro
blue tetrazolium (NBT) [22,30]. The reaction system contained
0.2 mM xanthine and 0.6 mM NBT in 0.1 M phosphate buffer pH 7.8.
The tested compounds were dissolved in DMF and the final
concentration of DMF in the reaction mixture was 1% in 0.1 M
phosphate buffer pH 7.8. The measured concentrations of the
compounds were 0.5 μM, 1.0 μM, 1.5 μM, 2.0, μΜ, 2.5 μM, 4 μM,
5 μM, 7.5 μM and 10 μM. 0.07 U/mL, xanthine oxidase was added to
the reaction to start. Each experiment was performed in triplicate.
4½CuðmecloÞ2ðH2OÞ2ꢀ þ 4DMSO→½Cu4ðmecloÞ6ðOHÞ2ðDMSOÞ2ꢀ•2DMSO
þ2Hmeclo þ 6H2O
Recrystallization of [Cd(meclo)2(H2O)2] (5) from DMSO solution
gives the complex [Cd(meclo)2(DMSO)3] (5a). The coordinated
molecules of water are replaced by the coordinated molecules of
DMSO.
3. Results and discussion
½CdðmecloÞ2ðH2OÞ2ꢀ þ 3DMSO→½CdðmecloÞ2ðDMSOÞ3ꢀ þ 2H2O
3.1. Synthetic aspects
The complexes are microcrystalline or powder-like and stable in
atmospheric conditions. All the complexes are insoluble in water,
except of 5 which is slightly soluble in water. Complexes 2 and 3 are
soluble in the solvents Me2CO, CHCl3, CH2Cl2, EtOH, DMSO, DMF, THF,
benzene, hexane, pentene and toluene. Complexes 4 and 5 are soluble
in DMSO, DMF and slightly soluble in THF, CH2Cl2 and CHCl3. The
elemental analyses confirm their stoichiometry. The μeff values of
complexes 2 and 3show that these are high spin. The observed values
of μeff=5.66 and μeff=1.80 BM for complexes 2 and 3 respectively
are typical for mononuclear Mn(II) and Cu(II) complexes with d5 and
Compound 1 was synthesized by the condensation of (2-
carboxyphenyl)phenyliodonium, with 2,6-dichloro-3-methylbenze-
namine in the presence of anhydrous copper(II) acetate as described
in the literature [19]. Compounds 2–5 were obtained by the aerobic
reaction of metal(II) acetates Mn(II), Cu(II) and Zn(II) or of Cd(II)
chloride in the presence of drops of triethylamine till the apparent pH
value was 7. The molar ratio for all the reactions (reactions (1–3) was
1:2 M/L.
d
9 configuration (S=5/2) and (S=1/2 respectively.
½MnðCH3COOÞ2ꢀ þ Hmeclo→½MnðmecloÞ2ꢀ þ 2CH3COOH
ð1Þ

D. Kovala-Demertzi et al. / Journal of Inorganic Biochemistry 105 (2011) 1187–1195
1191
3.2. Spectroscopy
that complexes 2 and 3 are solvated with two solvent molecules
coordinating in a pseudo-octahedral arrangement. The same pattern
of spectrum is shown when compounds 2 and 3 are dissolved in DMF
or CHCl3. It is suggested that these solvent molecules are exchanged
with the axial ligands of 3 (water molecules) [10,12].
3.2.1. Infrared spectroscopy
As the carboxyl hydrogen is more acidic than the amino hydrogen
the deprotonation occurs in the carboxylic group. This is confirmed by
the IR spectra of the complexes, showing the characteristic bands for
the secondary amino groups and for the coordinated carboxylato
group. A broad absorption at 3400–3300 cm−1 in the spectra of the
complexes was attributed to the presence of coordinated water. The
absence of large systematic shifts of the v(NH) and δ(NH) bands in the
spectra of the complexes compared with those of the ligand indicates
that there is no interaction between the NH group and the metal ions.
The νas(COO) and νsym(COO) bands of the prepared complexes are
at 1615–1618 and at 1390–1397 cm−1 respectively; the difference
Δ [νas(COO)−νsym(COO)] between these frequencies for 2–5 is
~220 cm−1, which is close to that found for the anisobidentate
chelate carboxylato group. The medium bands at ~480–430 cm−1
is attributed to the v(M-OH2O) stretching mode, while the bands at
370–250 cm−1 to the v(M-Ooco) stretching mode [7,8].
3.2.3. NMR spectra
Peak assignments were based on 2D NMR data {proton–proton
correlated spectroscopy (1H–1H COSY), proton–carbon heteronuclear
single-quantum coherence (1H–13C HSQC) and proton–carbon het-
eronuclear multiple-bond correlation (1H–13C HMBC). The downfield
chemical shift for HN in the spectra of ligand indicates that this proton
is involved in hydrogen bonding. The existence of the HN resonance in
the 1H-NMR spectra of complexes indicates that the nitrogen atom
remain protonated and the downfield chemical shift for this group
indicates that this proton is involved in an intramolecular hydrogen
bond between the HN group and the carbonyl oxygen of the
carboxylato group. In the 1H and 13C NMR spectra of the Zn(II) and
Cd(II) complexes 4 and 5 the hydrogen and carbon atoms of the
phenyl (C1–C6) ring were shifted upon coordination, which indicated
variations in the electron density of the phenyl ring. Deshielding of
H(3) and C(3) is observed in complexes, which should be related to
the electrophilicity of the metal centers. A σ-charge donation from the
COO− donor to the metal center removes electron density from the
ligand and produces this deshielding. Involvement of the carboxyl
group in bonding to Zn(II) and Cd(II) is confirmed by the resonances
ascribed to carboxyl carbon, which exhibit the greatest shifts upon
coordination. The 13C-NMR spectra of the complexes reveal major
changes in the exocyclic carboxylic group, indicative of metal
interaction through the carboxylic group. The latter exhibits either a
3.2.2. Electronic spectroscopy
The electronic spectra of 2 and 3 were recorded in DMF and CHCl3
solution. The electronic spectra of the Mn(II) complex, 2, can be
assigned six coordinate stereochemistry [7,8]. The absorption of the
organic ligand tailing into the visible region obscures the very weak
d–d absorption bands of the manganese(II) complex. The broad
visible band envelope of 3 will contain dxy→dx2−y2 and dxz,
dyz→dx2−y2 at six-coordinated tetragonally distorted stereochem-
istry. The d–d spectra of the solvated 2 and 3 can be assigned to
transitions in pseudo-octahedral structures or six-coordinated tetrag-
onally distorted stereochemistries. The solution spectral data indicate
Fig. 1. Labelled ORTEP diagram of 3a.

1192
D. Kovala-Demertzi et al. / Journal of Inorganic Biochemistry 105 (2011) 1187–1195
major downfield shift. The remaining resonances due to the aromatic
carbon atoms do not shift significantly on binding to Zn(II) and Cd(II).
Analysis of the shape determining angles for Cu(1), using the approach
of Reedijk and coworkers [31], yields τ ((α−β)/60) a value of 0.14 for
Cu(1) (τ=0.0 and 1.0 for square–pyramidal (SP) and trigonal–
bipyramidal (TBP) geometries respectively). The metal coordination
geometry is therefore described as distorted square pyramidal for Cu(1)
with the O(10) atom occupying the apical position for Cu(1). The donor
O(10) is chosen as apex by the simple criterion that it should not be one
of the oxygens which define either of the two largest L–Cu–L angles, α
and β [29]. The metal coordination geometry is described as distorted
octahedral for Cu(2). The phenyl rings are planar. The dihedral angles
between the planes of the phenyl rings are 66.6(3) and 61.1(6)° for the
bidentate bridging the Cu(1)–Cu(2) and Cu(2)–Cu(1a) metal centers
respectively. The dihedral angle for the anisobidentate chelating ligand
is 53.3(3)°, while in meclofenamic acid, the two planes are virtually
perpendicular, the corresponding angle being 81° [32]. The amino-
benzoate portion of each carboxylato ligand is effectively planar
which facilitates the formation of intramolecular N(17)–H…O(12),
N(37)–H…O(32) and N(57)–H…O(52) interactions of 2.669(5),
2.606(5) and 2.698(5) Å, respectively. The bidentate bridging
meclofenamato has a difference of 0.011 and 0.028 Å (O(11)–C(10)
1.272(5), O(12)–C(10) 1.261(5); O(51)–C(50) 1.252(5), O(52)–C(50)
1.280(5) Å) between its C–O bonds while for the anisobidentate
chelated meclofenamato this difference is 0.045 Å (O(31)–C(30)
1.289(5) O(32)–C(30) 1.244(5) Å); the variations in the C–O bond
distances suggest charge delocalization over the carboxylato group COO.
The different modes of bonding of the acetates, i.e. bridging or chelating,
are thus easily differentiated by the relevant bond lengths. Intra and
intermolecular hydrogen bonds, π–π and C−H−–π interactions
stabilize this structure, Table 3.
3.3. Crystal structures
3.3.1. Crystal structure of 3a
A diagram of 3a is shown in Fig. 1, and selected bond distances and
angles are listed in Table 2. Compound 3a is a centrosymmetric tetramer
built up around the planar cyclic Cu2(OH)2 unit. The two oxygen atoms
of this unit are linked in a μ3-O fashion through three Cu atoms, two
endo-cyclic and one exo-cyclic. The two endo and the two exo-cyclic Cu
atoms are symmetrically bridged by four meclofenamato ligands
through the carboxylato oxygens (Cu(1)–O(52) 1.929(3) and Cu(2)–
O(51) 1.990(3) and Cu(1)–O(11) 1.934(3) and Cu(2)–O(12) 1.991(3)
Å). Additional links between the endo- and exo-cyclic Cu atoms are
provided by two DMSO molecules that are linked in a μ2-O fashion
and symmetrically bridge two metal centers (Cu(1)–O(10) 2.266(3)
and Cu(2)–O(10) 2.277(3) Å). Each exocyclic Cu atom is also
coordinated by an anisobidentate chelating meclofenamato ligand
(Cu(1)–O(31) 1.936(3) Å and Cu(2)–O(32) 2.684(3) Å). The distance
between the endocyclic and exocyclic Cu atoms is 3.0987(7) Å, the
distance between the two endocyclic Cu centers is 2.9045(10) Å, and the
distance between the two exocyclic Cu centers is 3.3276(7) Å. The
coordination number is five and six for Cu(1) and Cu(2), respectively.
Table 2
Selected bond distances (A°) and angles (°) for 3a and 5a.
[Cu4(meclo)6(OH)2(DMSO)2]•2DMSO (3a)
[Cd(meclo)2(DMSO)3] (5a)
Cu(1)–O(52)#1
Cu(1)–O(11)
Cu(1)–O(1)#1
Cu(1)–O(1)
Cu(1)–O(10)
Cu(1)–Cu(1)#1
Cu(1)–Cu(2)
Cu(1)–Cu(2)#1
Cu(2)–O(31)
Cu(2)–O(1)
Cu(2)–O(51)
Cu(2)–O(12)
Cu(2)–O(10)
Cu(2)–O(32)
O(1)–Cu(1)#1
O(52)#1–Cu(1)–O(11)
O(52)#1–Cu(1)–O(1)#1
O(11)–Cu(1)–O(1)#1
O(52)#1–Cu(1)–O(1)
O(11)–Cu(1)–O(1)
O(1)#1–Cu(1)–O(1)
O(52)#1–Cu(1)–O(10)
O(11)–Cu(1)–O(10)
O(1)#1–Cu(1)–O(10)
O(1)–Cu(1)–O(10)
O(31)–Cu(2)–O(1)
O(31)–Cu(2)–O(51)
O(1)–Cu(2)–O(51)
O(31)–Cu(2)–O(12)
O(1)–Cu(2)–O(12)
O(51)–Cu(2)–O(12)
O(31)–Cu(2)–O(10)
O(1)–Cu(2)–O(10)
O(51)–Cu(2)–O(10)
O(12)–Cu(2)–O(10)
O(31)–Cu(2)–O(32)
O(1)–Cu(2)–O(32)
O(51)–Cu(2)–O(32)
O(12)–Cu(2)–O(32)
O(10)–Cu(2)–O(32)
Cu(2)–O(1)–Cu(1)#1
Cu(2)–O(1)–Cu(1)
Cu(1)#1-O(1)–Cu(1)
Cu(1)–O(10)–Cu(2)
1.929(3)
1.934(3)
1.959(3)
1.960(3)
2.266(3)
2.9045(10)
3.0987(7)
3.3278(7)
1.936(3)
1.944(3)
1.990(3)
Cd(1)–O(31)
Cd(1)–O(11)
Cd(1)–O(3)
Cd(1)–O(12)
Cd(1)–O(1)
Cd(1)–O(2)
Cd(1)–O(32)
2.270(3)
3.3.2. Crystal structure of 5a
2.352(3)
2.376(4)
2.377(4)
2.389(4)
2.396(4)
2.418(4)
A diagram of 5a is shown in Fig. 2. The complex is mononuclear with
the meclofenamato ligand behaving as a bidentate deprotonated
chelating ligand [Cd–O(11) 2.352(3), Cd–O(12) 2.377(4) and Cd–O(31)
2.270(3), and Cd–O(32) 2.418(4) respectively for the two ligands],
coordinated to cadmium atom via a carboxylate oxygen atoms. The
three DMSO molecules are coordinated to cadmium atom via the
oxygen atom [Cd–O(1) 2.389(4), Cd–O(2) 2.396(4) and Cd–O(3)
2.376(4) for the three molecules of DMSO], thus rendering cadmium
atom seven-coordinated. All the Cd–O distances are of the same
magnitude with Cd–O(11)=2.353(3) being the shortest and the
Cd–O(32) 2.418(4) being the longest from the two carboxylato
groups. The dihedral angles between the planes of the phenyl rings
for 3a are 71.3(3)° and 65.4(3)° for the two bidentate chelating
ligands, respectively, while in meclofenamic acid, the two planes are
virtually perpendicular, the corresponding angle being 81° [32]. The
polar imino hydrogen atoms on N, participates in an intramolecular
hydrogen bond. Intra and intermolecular hydrogen bonds stabilize
the structure, while the crystal packing is determined by π–π and
C−H−–π interactions. Strong π–π interaction occur between the
phenyl rings C(18)–C(32) (symmetry operation; −x, 1−y, −z) at
a centre-to-centre separation of 3.767(4) and between the phenyl
rings C(38)–C(43) (symmetry operation; −x, 1−y, 1−z) at a
centre-to-centre separation of 3.808(3), Table 3.
1.991(3)
2.277(3)
2.684(3)
1.959(3)
86.28(13)
92.58(12)
175.78(12)
167.60(12)
95.92(12)
84.34(12)
113.24(11)
92.94(11)
91.24(11)
78.90(11)
176.11(12)
87.74(12)
93.18(12)
87.79(12)
92.38(12)
163.07(12)
97.18(11)
78.94(11)
106.36(11)
90.40(11)
54.43(11)
129.45(11)
75.50(11)
88.64(11)
151.61(10)
117.00(14)
105.08(12)
95.66(12)
86.02(10)
O(31)–Cd(1)–O(11)
O(31)–Cd(1)–O(3)
O(11)–Cd(1)–O(3)
O(31)–Cd(1)–O(12)
O(11)–Cd(1)–O(12)
O(3)–Cd(1)–O(12)
O(31)–Cd(1)–O(1)
O(11)–Cd(1)–O(1)
O(3)–Cd(1)–O(1)
O(12)–Cd(1)–O(1)
O(31)–Cd(1)–O(2)
O(11)–Cd(1)–O(2)
O(3)–Cd(1)–O(2)
O(12)–Cd(1)–O(2)
O(1)–Cd(1)–O(2)
O(31)–Cd(1)–O(32)
O(11)–Cd(1)–O(32)
O(3)–Cd(1)–O(32)
O(12)–Cd(1)–O(32)
O(1)–Cd(1)–O(32)
O(2)–Cd(1)–O(32)
159.88(13)
108.51(14)
78.27(14)
138.55(12)
55.51(12)
97.51(13)
82.83(13)
84.71(13)
156.67(15)
85.62(13)
79.63(14)
120.45(14)
74.59(17)
76.87(13)
128.35(15)
55.76(12)
108.53(12)
80.94(14)
163.67(12)
89.60(13)
117.86(13)
3.4. Pharmacology
3.4.1. Antiproliferative activity in vitro
The results of cytotoxic activity in vitro are expressed as IC50— the
concentration of compound (in μM) that inhibits a proliferation rate of
the tumor cells by 50% as compared to control untreated cells, Table 4.
Meclofenamic acid and the metal complexes 1–5 were tested for their
antiproliferative activity in vitro against the cells of three human
cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder
cancer cell line), A-549 (non-small cell lung carcinoma) and a mouse
fibroblast L-929 cell line and the results are compared with the known

D. Kovala-Demertzi et al. / Journal of Inorganic Biochemistry 105 (2011) 1187–1195
1193
Table 3
C−H−–π, π–π, inter- and intra- molecular hydrogen bonds for 3a and 5a; Cg(1) and Cg(2) refer to the centroids C(11)–C(12)–C(13)–C(14)–C(15)–C(16) and C(58)–C(59)–C(60)–
C(61)–C(62)–C(63) and Cg(3) refer to the centroid C(31)–C(32)–C(33)–C(34)–C(35)–C(36) for 3a. Cg(1), Cg(2) and Cg(3) refer to the centroids C(18)–C(19)–C(20)–C(21)–C(22)–
C(23), C(38)–C(39)–C(40)–C(41)–C(42)–C(43) and C(31)–C(32)–C(33)–C(34)–C(35)–C(36) resp., for 5a. D and Α refers to donor and acceptor respectively.
[Cu4(meclo)6(OH)2(DMSO)2]•2DMSO (3a)
Cg–Cgb
βc
CgI-Perpd
CgJ-Perpe
Cg(1)–NCg(2)vii
Cg(1)–NCg(2)vii
4.376(3)
4.376(3)
39.42
15.09
4.225(2)
3.380(3)
3.381(3)
4.225(2)
H—Cg
C—Cg
C–H—Cg
C(3)–H(3B)–NCg(3)viii
2.79
3.539(7)
120
D
H
Ab
D ⋯ A
H ⋯ A
D - H ⋯ A
N(17)
N(37)
O(1)x
C(3)ix
H(17A)
H(37A)
H(1)
O(12)
O(32)
O(20)
O(32)
2.669(5)
2.606(5)
2.639(4) 3.190(7)
2.05
1.80
1.90
2.54
123
143
177
127
H(3C)
[Cd(meclo)2(DMSO)3] (5a)
Cg–Cgb
βc
CgI-Perpd
CgJ-Perpe
Cg(1)NCg(1)ii
3.767(4)
3.808(3)
21.82
22.90
−3.497(3)
−3.497(3)
−3.497(3)
−3.508(2)
Cg(2)–NCg(2)iv
H—Cg
C—Cg
C–H—Cg
C(4)–H(4B)–NCg(3)v
C(14)–H(14)–NCg(1)vi
2.63
2.82
3.476(7)
3.655(7
138
142
D
H
Ab
D ⋯ A
H ⋯ A
D–H ⋯ A
N(17)
N(37)
C(1)i
H(17A)
H(37A)
H(1B)
H(24C)
H(33)
O(11)
O(31)
O(2)
O(32)
O(12)
O(1)
2.630(5)
2.630(5)
3.478(8)
3.440(9)
3.470(8)
3.404(6)
1.91
2.03
2.56
2.47
2.60
2.46
147
147
156
170
154
163
C(24)ii
C(33)iii
C(41)iv
H(41)
aSymmetrytransformations, i,=−1+x, y,z; ii, −x, 1−y, −z; iii, 1+x, −1+y,z;iv, −x, 1−y,1−z; v, −x, 1+y,z;vi, −1−x, 2−y, −z; vii, 2−x, 1−y,1−z;viii, ½+x, ½−y, −1/2+z;
ix, −1/2+x, 1/2−y, −1/2+z; x, 1/2+x, 1/2−y,1/2+z; bCg–Cg is the distance between ring centroids; cWhere β is the angle Cg(I)–-NCg(J); dCgI-Perp is the perpendicular distance
of Cg(I) on ring J; eCgJ-Perp is the perpendicular distance of Cg(J) on ring I.
chemotherapeutic cis-platin and related complexes of tolfenamic acid
and mefenamic acid [7,8]. Meclofenamic acid exhibits poor cytotoxic
activity against MCF-7 and T24 cell lines and very poor cytotoxic
activity against L-929 and A-549 cell lines. Metal salts are inactive
against L-929, A-549 and MCF-7 and exhibit very poor activity against
T-24 cancer cell lines. Also, complex 4 is inactive against A-549 cancer
cell line.
complexes 2–5 range from 5 to 40 μM against MCF-7 and 3–5 μM
against T-24 cancer cell while for cis-platin the IC50 values range from
1 to 42 μM against all cancer cell lines tested. Complexes 2–5 are less
cytotoxic than cis-platin against L-929 ans A-549 cancer cell lines.
Complexes 2–4 are in the same range as cis-platin, 4–5 times less
cytotoxic than cis-platin while complex 5 is 1.6 times more cytotoxic
than cis-platin. Complexes 2–5 are 8–13 times more cytotoxic than
cis-platin against T-24 cancer cell lines. Selectivity was observed for
complexes 4 and 5 against T-24 and for complex 5 against MCF-7
The IC50 values for complexes 2–5 range from 23–62 μM against
L-929 and A-549 cancer cell lines tested. The IC50 values for
Fig. 2. Labelled ORTEP diagram of 5a.

1194
D. Kovala-Demertzi et al. / Journal of Inorganic Biochemistry 105 (2011) 1187–1195
Table 4
results for a number of Cu(II)-NSAIDs using NBT assay were investigated
[6,18]. Complexes exhibited SODactivitywith IC50 values ranging from 1
to 28 μM. Very low values for a manganese superoxide dismutase mimic
were reported by Nagano and coworkers (0.75 μM) and Rajan (0.75 μM)
[33,34]. In both of these complexes manganese is in the divalent form.
However, in dismutases containing manganese the metal is normally in
the trivalent state, but can be reduced to the divalent state without any
loss of enzymic activity. It is also worth noting that many of the high
activity complexes are neutral rather than anionic. Complexes 2 and
3show an IC50 value of 6.48 0.81 and 1.78 0.16 (μM) respectively,
while the parent drug meclofenamic and the complexes of Zn(II) and Cd
(II) did not show any SOD activity. These values indicate that 3 is a
potent superoxide dismutase mimic. Similar results have been obtained
for copper(II) complexes [6,18]. It was found that the IC50 values for the
corresponding copper(II) complexes of tolfenamic and diclofenac [Cu
(tolf)2(H2O)]2 and [Cu(dicl)2(H2O)]2•2H2O are 1.97 0.17 and 2.13
0.11(μM) respectively [16]. Among the copper(II) fenamates [18]
complex 3 exhibits the highest SOD activity. Following the usual criteria
advanced by Roberts and Robinson [35] the present copper(II) complex
is considered as potent SOD mimic.
The antiproliferative activity in vitro of 1–5, related NSAIDs and metal complexes
(expressed as IC50 (μM) against MCF-7, T-24, A-549 and L-929 cancer cell lines).
MCF-7
T-24
70.2 0.3
3.8 1.3
5.3 0.6
3.2 1.0
3.3 1.6
A-549
L-929
HMeclo (1)
[Mn(meclo)2] (2)
[Cu(meclo)2(H2O)2] (3) 38.2 0.9
63.1 0.4
39.8 2.4
139.1 0.7 133.0 0.8
27.4 3.2
41.2 5.6
25.3 2.4
61.9 3.9
24.2 2.8
22.7 2.2
214 18
149
5.3 0.5
123
d
[Zn(meclo)2(H2O)2](4)
[Cd(meclo)2(H2O)2](5)
HTolfa
32.5 2.1
5.1 1.2
87.9 6.3
41.6 2.9
26.1 2.1
41.3 3.5
149.2 3.2
72.6 3.5
25.1 3.3
40.7 5.0
n.o.c
N244
29.8 1.4
145 12
65.3 6.1
31.4 2.2
57.9 4.2
62.4 5.1
3.9 0.3
[Mn(tolf)2(H2O)2]a
6
a
[Cu(tolf)2(H2O)]2
13. 9 1.1
41.0 2.5
81.2 4.5
35.1 1.0
7.77 2.2
37.7 2.0
53.4 9.6
50.1 10.0 n.o.
95.7 16.2 n.o.
130.7 7.0
41.7 4.5
[Zn(tolf)2(H2O)]a
Hmefb
6
168.3 2.3 178.2 2.8
[Mn(mef)2(H2O)2]b
N175.6d
N175.6
19.5 3.0
d
b
d
[Cu(mef)2(H2O)]2
N100.3
111.2 3.0 74.4 1.0
n.o.
[Zn(mef)2]b
Mn(Ac)2
n.o.
233.7 15.3
n.o.
n.o.
0.7 0.1
Cu(Ac)2 H2O
Zn(Ac)2 2H2O
CdCl2 2H2O
Cis-platin
n.o.
n.o.
n.o.
8.0 0.8
n.o.
1.5 0.1
a
Data taken from ref. [7].
Data taken from ref. [8].
Activity not observed.
b
c
Acknowledgments
d
The IC50 value is not possible to be calculated, since the inhibition for the higher
concentration (100 μg/mL) was found to be 30–35%. In that case, the IC50 value will be
higher than the concentration of 244 μM for the complex [Zn(meclo)2(H2O)2](4) or
higher than the concentration of 175.6 μM for the complex [Mn(mef)2(H2O)2].
G.S. thanks IKY for a scholarship for her Ph.D. We thank Dr. P.
Stathopoulos for low resolution mass spectra obtained on a machine
funded by the Network of Research Supporting Laboratories of the
University of Ioannina. We thank the NMR service of the University of
Ioannina, Greece.
cancer cell lines. Also, it was found that the metal complexes of
tolfenamic acid (Htolf), [Mn(tolf)2(H2O)2] and [Cu(tolf)2(H2O)]2
have shown selectivity against T-24 cell line [7,8]. Compounds 2–5
exhibited high activity as anticancer agents against T-24 cancer cell
lines, and exhibited selectivity against the same cancer cell line.
Compound 5 exhibits the highest selectivity against MCF-7 and 4
shows the highest selectivity against T-24. From the in vitro
experimental results, it is indicated that meclofenamic acid and the
metal acetates are non active or less active than their corresponding
metal complexes of meclofenamic, which indicates that the mech-
anism of action is different.
The reported therapeutic action of these metal complexes could be
either due to a small proportion of metal complexes that can bind
intact to biomolecules and can be transported to the site of action, or
to a ternary Metal-meclofenamic-biomolecule complex which is also
the transporting agent [7]. Interestingly enough, 2–5 were found to be
more potent cytotoxic agents against T-24 and 5 the most potent
agents against MCF-7 cancer cell lines than the prevalent benchmark
metallodrug, cis-platin, under the same experimental conditions
measured by us and this assumes significance in light of the fact
that cis-platin is undisputedly the most studied and widely used
metallopharmaceutical for cancer therapy known to date.
Appendix A. Supplementary data
Crystallographic data for the structural analysis has been
deposited with the Cambridge Crystallographic Data Centre, CCDC
814419 and 814420 for compounds 3a and 5a respectively. Copies
of this information may be obtained free of charge from The
Director, CCDC, 12, Union Road, Cambridge CB2 1EZ [Fax: +44 1223
336 033] or e-mail deposit@ccdc.cam.ac.uk or http://www.ccdc.
cam.ac.uk. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bbapap.2011.06.003.
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