Thermal, spectroscopic and biological studies on solid ibuprofen complexes of heavy trivalent lanthanides and yttrium

Article abstract of DOI:10.1016/j.tca.2016.11.007

Heavy lanthanide complexes (Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) and yttrium (III) complexes with ibuprofen ligands (Hibu) were synthesized and characterized by simultaneous thermogravimetric and differential thermal analysis (TG-DTA), differential scanning calorimetry (DSC), attenuated total reflection mid-infrared spectroscopy (ATR-MIR), complexometric titration, X-ray powder diffraction (XRD) in order to determine stoichiometry, thermal stability and the ligand coordination modes of the compounds. The volatiles released were also analyzed by online coupled thermogravimetry-infrared spectroscopy evolved gas analysis (TG-EGA-MIR), to identify the main product resulting from the heating of terbium complex. In addition, ibuprofen and the synthesized compounds were tested to assess cytotoxic/proliferative and anti-inflammatory activity. The results of the cytotoxicity assays showed that compounds [Yb(ibu)₃], [Lu(ibu)₃] and [Y(ibu)₃] decreased the cytotoxic activity of ibuprofen. Furthermore, [Yb(ibu)₃] and [Lu(ibu)₃] exhibited a significant anti-inflammatory profile, superior to that of ibuprofen. Under the stimulatory effect of lipopolysaccharide, these compounds displayed anti-inflammatory activity characterized by low TNF-α and H₂O₂ production and high IL-10 production, emerging as interesting alternatives for further biological applications.

Full text of DOI:10.1016/j.tca.2016.11.007

Accepted Manuscript
Title: Thermal, spectroscopic and biological studies on solid
ibuprofen complexes of heavy trivalent lanthanides and
yttrium
Author: Bruno B.C. Holanda Renan B. Guerra Alexandre O.
ˆ
Legendre D e´ bora F. Almeida Thais F.C. Fraga-Silva Angela
C. Finato James Venturini Gilbert Bannach
PII:
S0040-6031(16)30308-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.tca.2016.11.007
TCA 77635
To appear in:
Thermochimica Acta
Received date:
Revised date:
Accepted date:
5-10-2016
8-11-2016
9-11-2016
Please cite this article as: Bruno B.C.Holanda, Renan B.Guerra, Alexandre
ˆ
O.Legendre, D e´ bora F.Almeida, Thais F.C.Fraga-Silva, Angela C.Finato, James
Venturini, Gilbert Bannach, Thermal, spectroscopic and biological studies on solid
ibuprofen complexes of heavy trivalent lanthanides and yttrium, Thermochimica Acta
http://dx.doi.org/10.1016/j.tca.2016.11.007
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Thermal, spectroscopic and biological studies on solid
ibuprofen complexes of heavy trivalent lanthanides and
yttrium
1
1
1
2
Bruno B. C. Holanda , Renan B. Guerra , Alexandre O. Legendre , Débora F. Almeida ,
2
2
2
1*
Thais F. C. Fraga-Silva , Ângela C. Finato , James Venturini , Gilbert Bannach
1
Faculdade de Ciências, Departamento de Química, UNESP — Univ. Estadual Paulista, 17033-360 Bauru, São Paulo,
Brasil.
2
Faculdade de Ciências, Departamento de Ciências Biológicas, UNESP — Univ. Estadual Paulista, 17033-360 Bauru,
São Paulo, Brasil.
*gilbert@fc.unesp.br
HIGHLIGHTS
•
•
•
•
Ibuprofen complexes of heavy trivalent lanthanides and yttrium have been synthetized.
The thermal analysis provided information about the thermal behavior of the samples.
Analysis of the IR spectra showed two coordination modes for compounds.
The TG-EGA-MIR showed propane as the gas evolved during the thermal
decomposition.
Cytotoxicity and anti-inflammatory activity of the compounds were evaluated.
•
Abstract
Heavy lanthanide complexes (Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) and yttrium (III) complexes with ibuprofen ligands
(
Hibu) were synthesized and characterized by simultaneous thermogravimetric and differential thermal analysis (TG-
DTA), differential scanning calorimetry (DSC), attenuated total reflection mid-infrared spectroscopy (ATR-MIR),
complexometric titration, X-ray powder diffraction (XRD) in order to determine stoichiometry, thermal stability and the
ligand coordination modes of the compounds. The volatiles released were also analyzed by online coupled
thermogravimetry–infrared spectroscopy evolved gas analysis (TG-EGA-MIR), to identify the main product resulting
from the heating of terbium complex. In addition, ibuprofen and the synthesized compounds were tested to assess
cytotoxic/proliferative and anti-inflammatory activity. The results of the cytotoxicity assays showed that compounds
[
[
Yb(ibu)
Lu(ibu)
3
], [Lu(ibu)
3 3 3
] and [Y(ibu) ] decreased the cytotoxic activity of ibuprofen. Furthermore, [Yb(ibu) ] and
3
] exhibited a significant anti-inflammatory profile, superior to that of ibuprofen. Under the stimulatory effect
2 2
of lipopolysaccharide, these compounds displayed anti-inflammatory activity characterized by low TNF-α and H O
production and high IL-10 production, emerging as interesting alternatives for further biological applications.
Keywords: thermal behavior, middle infrared spectroscopy, TG-EGA-MIR, metallodrug, lanthanides, biological studies
1
. Introduction
1

The literature reflects significant interest in the study of compounds containing metals, given their potential as drugs
metallodrugs) or diagnostic agents as well as the need to understand their mechanisms of action [1]. Since the
(
discovery of inhibitory effect of cisplatin on cell proliferation and its application in chemotherapy, scientific interest in
developing new metal-based drugs has grown [2,3]. In addition to their important pharmacological properties,
metallodrugs may also exhibit different biodistribution profiles, influenced by both the nature of the metal center and
the physicochemical properties of the ligands [4,5]. Many compounds with potential as metallodrugs are limited or
unsuccessful due to the adverse effects of intrinsic resistance mechanisms acquired by the disease [6-8].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to combat symptoms and illnesses such as pain
and inflammatory processes. However, side effects in the gastrointestinal system mean their use is restricted [9, 10].
Ibuprofen (Hibu), whose chemical structure is represented in Fig. 1, belongs to this group of anti-inflammatories,
specifically the subgroup of propionic acid derivatives, which have a common ability to reduce inflammation, pain and
fever [11].
Thermoanalytical techniques are widely used in the pharmaceutical industry, quality control sector and research
laboratories to study physical and chemical phenomena, such as thermal decomposition, thermal stability [12-14],
polymorphism [15, 16], solid state reactions, drug formulation [17-19], purity [20], reaction kinetics [21], analysis of
the volatile products released coupled with TG-EGA-MIR analysis [22, 23], as well as other properties of solid
compounds used in the pharmaceutical industry.
Despite exhibiting anti-inflammatory action, some NSAIDs such as ibuprofen and indomethacin increase the
production of inflammatory mediators, including TNF-α and IL-6 [24, 25]. The production of inflammatory metabolites
is essential to combat pathogens; however, an exacerbated inflammatory response can be detrimental, causing tissue
2 2
damage owing to high TNF-α levels [26], in addition to damaging DNA due to high H O production [27].
The motivation behind preparing lanthanide complexes with ibuprofen is based on their structural similarity with
other lanthanide complexes reported in the literature [28], which show potential in pharmacological, diagnostic and
therapeutic applications [29]. Thus, the present study aims to provide important information for future biological
applications. This article is a continuation of the reference study [30].
2
. Materials and methods
2
.1 Synthesis of complexes
Ibuprofen (acid form, Hibu) was purchased from Aldrich with ≥ 98% purity. The compounds were prepared
3
according to the procedure described below. Aqueous solutions of each lanthanide chloride (LnCl ) were prepared by
reacting approximately 0.200 g (1.40 mmol) of the corresponding metal oxide (Aldrich) with around 3.0 mL of
concentrated hydrochloric acid (Merck). The excess acid was eliminated via several evaporation cycles and the addition
of distilled water. Next, an ibuprofen sodium salt (Naibu) solution was prepared by adding an aqueous solution 0.10
-
1
mol L sodium hydroxide to 2.000 g (9.690 mmol) of ibuprofen suspension in distilled water, under stirring. The LnCl
3
and Naibu solutions were stirred together slowly, with precipitation of white solids, which were filtered through
Whatman 42 filter paper and washed with cold water to remove excess of chloride ions. A qualitative test was
conducted with AgNO
3
solution to confirm chloride removal. Finally, the precipitates were collected and stored in a
desiccator under reduced pressure and calcium chloride as desiccant agent.
2

2
.2 Instrumentation
Simultaneous TG-DTA and DSC curves were collected using a SDT 2960 and a DSC Q10 thermal analysis system
respectively, both from TA Instruments. The purge gas was air flow rate of 100 mL min−1 for TG-DTA and 50 mL
min−1 for DSC experiments. A heating rate of 20 ºC min−1 was adopted, with samples masses of approximately 5 mg for
TG-DTA and 3 mg for DSC runs. α-Alumina and aluminum crucibles, the latter with perforated cover, were used for
TG-DTA and DSC, respectively.
Attenuated total reflectance middle-infrared (ATR-MIR) spectra measurements were obtained with a Nicolet MIR
–
1
iS10 spectrophotometer using an ATR accessory and Ge window in the 4000 to 600 cm range.
Coupled analyses (TG-EGA-MIR) were carried out by connecting the exhaust of a TG–DSC (TGA/DSC Stare
System) system to a Nicolet iS10 spectrophotometer (Thermo Scientific), with a gas cell operating at 250 ºC and DTGS
(
deuterated triglycine sulfate) detector. The furnace and heated gas cell (250 ºC), both purged with nitrogen (50 mL
-
1
min ), were coupled via a heated 120 cm stainless steel transfer line measuring 3.0 mm in diameter. The MIR spectra
-
1
–1
were recorded using 32 scans per spectrum at a resolution of 4 cm , heating rate of 10 ºC min and approximate
sample mass of 20 mg.
Metal contents in each complex were determined by complexometric titration with EDTA solution in a piston
–2
burette and accuracy of 110 mL, using xylenol orange as indicator [31].
Powder X-ray diffraction measurements was carried out at room temperature using a Rigaku D/MAX-2100PC
diffractometer, with CuKα radiation (λ = 1.5405Å), and an Ni filter to eliminate Kβ radiation, a slit size of 10 mm was
used, with a 20 mA current, operating voltage of 40 kV and scan range between 10º and 80º.
2
2
.3 Biological Assays
.3.1 Obtaining peripheral blood mononuclear cells (PBMC)
The sample consisted of ten healthy subjects recruited from a blood bank. The inclusion criterion applied was the
signing of an informed consent form. The Research Ethics Committee of the Faculty of Sciences of UNESP Bauru, in
São Paulo State, Brazil, approved this study. All participants gave their informed consent for participation in the study
and publication of data. Peripheral blood was obtained from the participants by venous puncture and placed in 10 mL
sterile tubes containing 20 U mL-1 heparin. Mononuclear cells were recovered by density gradient separation using
Histopaque®-1077 (Sigma-Aldrich, St. Louis, MO, USA) and then washed with RPMI 1640 culture medium containing
streptomycin-penicillin, HEPES and L-glutamine (Nutricell, Campinas-SP, Brazil) for 10 min at 1500 rpm. Next, the
cells were resuspended in RPMI 1640 culture medium supplemented with 20% autologous serum (complete RPMI).
The cell count was done with Turk and the viability of PBMC was assessed according to Trypan Blue uptake of 0.1%.
-
1
The cell concentration was adjusted to 106 cells mL .
2
.3.2 Cytotoxicity assay
3

MTT (3-(4,5-dimetietiazol-2yl)-2,5-diphenyltetrazolium bromide) is a water soluble salt, which is reacted with
mitochondrial dehydrogenases present only in metabolically active cells (i.e., living cells) to produce water insoluble
purple formazan crystals [32]. This salt is therefore used to determine cell viability.
The compounds were dissolved by two-fold serial dilutions in 100% dimethyl sulfoxide (DMSO) and prepared
-
1
-1
using a concentration range from 1.56 mg mL to 50 mg mL . Were seeded into 96-well culture plates and stimulated
-
1
with different concentrations of each compound (1 µL pool to be tested and incubated for 96 hours at 37ºC in 5% CO
2
.
After incubation, the culture plate was centrifuged for 5 minutes at 1500 rpm. The supernatant was discarded and the
-
1
cells resuspended in complete RPMI medium containing MTT (5 mg mL ).
The plate was incubated for 2 hours at 37ºC in 5% CO and then centrifuged for 5 minutes at 1500 rpm. The
2
supernatant was removed and the cells resuspended in 100 µL of dimethyl sulfoxide (DMSO) per well. After 5 minutes,
the plate was read in an ELISA reader at 540 nm. The cytotoxicity index (CI) was calculated from the ratio of the test
culture stimulated by the compounds and a control culture containing only cells. Compounds that exhibited lower CI
values than those than those obtained in control cultures were considered cytotoxic, while those with higher values in
relation to controls were considered capable of increasing cell viability.
2
.3.3 Monocyte culture
Mononuclear cells were obtained as described in the section 2.3.1. To obtain human monocytes, mononuclear cells
6
-1
were counted and adjusted with phenol red at a concentration of 110 cells mL . Cells were seeded into a 96-well plate
and incubated for 1 hour at 37ºC under 5% CO . After this period, the supernatant was removed and adhered cells were
resuspended in complete RPMI. In order to evaluate the influence of compounds in an inflammatory environment,
2
-
1
monocytes underwent four treatments: 1) simultaneous stimulation with 10 mg mL lipopolysaccharide (LPS) and 1μL
pool-1 of compounds at a concentration of IC50, 2) LPS; 3) compound; and 4) non-stimulated culture. Cells were
incubated at 37ºC under 5% CO
2
. After 24 hours, culture supernatants were collected for cytokine quantification assay
and adherent cells were used to determinate of hydrogen peroxide production (H
2
O
2
).
2
.3.4 Hydrogen peroxide production (H
2
O )
2
The determination of hydrogen peroxide was carried out according to the methods described by [33]. The reading
was performed in microplate reader with 620 nm filter (EL800, BIO-TEK Instruments, Inc.). H concentration in the
samples was calculated, as an analytical curve (0.5 to 8.0 nM H ).
2
O
2
2 2
O
2
.3.5 Cytokines quantification
IL-10 and TNF-α levels were measured in the cell-free supernatants of the monocyte culture using a cytokine Duo-
Set Kit (DuoSet kit R & D Systems), according to the manufacturer’s instructions. Results were expressed in pg mL-1
based on the standard curve established for each test.
2
.3.6 Statistical analysis
4

The two matched groups were compared using the paired t-test. All statistical analyses were carried out using
GraphPad Prism 5.0 software (GraphPad Software. Inc., San Diego, California, USA) at a significance level of p <0.05
[
34]. Linear regression was performed to determinate IC50 using Excel 2010 (Microsoft Office).
3
. Results and discussion
3
.1. Analytical results
Analytical and thermoanalytical (TG) data are shown in Table 1. All data exhibited excellent agreement, suggesting
that the compounds obtained were of high purity. These results made it possible to establish the stoichiometry of the
compounds, which was in agreement with the general formula [Ln(ibu) ]·nH O, in which Ln represents trivalent
lanthanide ions (from Gd to Lu and Y), and n = 1.0 (Gd), 0.5 (Tb) and 0 (Dy to Lu and Y).
3
2
3
.2 Thermal analysis
3 2 3 3
The TG-DTA curves of the compounds [Tb(ibu) ]∙0.5H O, [Ho(ibu) ], [Lu(ibu) ] (representative of the other
synthesized compounds) are shown in Fig. 2. Initial mass loss is attributed to loosely bound water (adsorbed), since
dehydration occurs at low temperatures. The thermoanalytical curves indicate similar thermal stability for the anhydrous
compounds and that of the dehydrated ones after water loss are very similar within a range of 151 to 175 ºC. The TG-
DTA curves demonstrate that thermal decomposition of the anhydrous compounds occurs in four consecutive and/or
overlapping steps. The first and fourth mass loss stages correspond to a pronounced exothermic peak in the DTA curve.
This represents the onset of thermal decomposition of the compound (first step) and combustion of the charred residue
2 3
formed in the previous steps (fourth step), which led to the formation of the metallic oxides Ln O for Gd, Dy to Lu and
Y and Tb . On the other hand, the thermal events associated with the intermediate steps are either absent or not
4 7
O
pronounced. This can be attributed to slow mass loss (second step) or simultaneous endothermic and exothermic
reactions (third step, significant mass loss). The DTG curves corroborate these observations, since they exhibit small
and/or broad signals with several peaks. Data on temperature ranges, mass loss percentages and temperature peaks are
summarized in Table 2.
The DSC curves shown in the supplementary material (Fig. 1S) are very similar for all compounds after dehydration
and indicate exothermal events associated with initial mass loss in the TG curves, with the following enthalpy values,
-
1
expressed in kJ mol : 249.98 [Gd(ibu)
3
]∙H
2
O; 176.94 [Tb(ibu)
3
]∙0.5H
2
O; 387.24 [Dy(ibu)
3 3
]; 199.16 [Ho(ibu) ]; 386.92
[
Er(ibu)
3
]; 288.09 [Tm(ibu)
3
]; 241.52 [Yb(ibu)
3
]; 160.67[Lu(ibu)
3
]; 189.89 [Y(ibu)
3
].
3
.3 Attenuated total reflectance and middle-infrared spectroscopy (ATR-MIR), TG-EGA-MIR and XRD
The infrared spectra of the sodium salt and the lanthanide complex [Tb(ibu)
synthesized compounds) are shown in Fig. 3 and the main bands in Table 3. Coordination in ibuprofen complexes
occurs through the carboxylate group, where the main bands in MIR spectra are corresponding to symmetrical ( ) and
) stretching, which comprises the region between around 1600 and 1400 cm . The coordination mode
3 2
]∙0,5H O (representative of the other
s
-
1
asymmetrical (
a
of the ligand in a complex can be determined by calculating the difference between the position of these bands (Δ = 
a
-
5

s
 ) [35]. When the coordination mode is bidentate, there is a little change in Δ when compared with the ionic bond,
whereas significantly low Δ values indicate chelation by the carboxylate group. In this research, MIR spectra for all the
complexes exhibited two bands in the region of the symmetric stretching frequency of the carboxylate group, which
suggests the existence of two coordination modes for this ligand and/or the presence of strong hydrogen bonds with
water molecules. Although asymmetric stretching frequencies for these compounds show only single bands, all of the
bands exhibit a shoulder, thus supporting the previous conclusion. However, Table 3 shows similar Δ values for
hydrated and anhydrous complexes, indicating that the band splitting must be the result of two coordination modes,
since hydrogen bond donors in deprotonated ibuprofen are not strong enough to produce split bands. The same
2 2 2 2
behaviour was observed for [Dy(2-MeO-Bz) µ-(2-MeO-Bz)(H O) ] , 2-MeO-Bz = 2-methoxybenzoate, whose structure
was determined by single-crystal X-ray diffraction [36, 37]. Thus, taking into account the Δ values from Table 3, it can
suggest that each complex contains chelating and bridging or bridging-chelating carboxylate groups [38]. The infrared
spectra of all the compounds are shown in the supplementary material (Fig. 2S).
Infrared spectra of the volatile products released during the first stage of compound decomposition were
measured and compared against databases [39], indicating that propane and other gaseous products were released
together, as shown in a previous study [30]. Spectra for pure propane and the released volatiles, represented here by
3 2
decomposition of [Tb(ibu) ]∙0.5H O, are shown in Fig. 4. The infrared spectra obtained for the first stage of thermal
decomposition of the anhydrous compounds are presented in the supplemental material (Fig. 3S).
The X-ray powder diffractograms demonstrate a low degree of crystallinity for the compounds obtained and no
evidence indicating the formation of an isomorphous series. All diffractograms are provided in the supplementary
material (Fig. 4S).
3
.4 Biological assay
The cell viability assay was carried out based on the reduction of MTT by mitochondrial enzymes of live cells. This
process results in the formation of purple crystals, called formazan crystals [32], where a large amount of crystals
indicates greater cell viability. As such, this assay makes it possible to determine the cytotoxicity mediated by the
substance to be tested. Thus, the results were analysed in two ways: the first related to index of citotoxity, indicating
whether they are toxic or induce cell viability and, the second assessment used the results of the first assessment to
identify the inhibitory concentration of 50% (IC50) of the cells.
3
.5 Cytotoxic activities
Cytotoxicity assay results are summarized in Fig. 5. An assessment of the different concentrations used indicated
-
1
that ibuprofen (ibu) had a cytotoxic effect at the initial concentration of 50 mg mL . On the other hand, concentrations
-
1
of 6.25, 3.12 and 1.56 mg mL increased cell viability. In general, we found that the compounds [Yb(ibu)
3
], [Lu(ibu)
] exhibited no
] showed higher cell viability at concentrations of 12.5; 6.25; 3.12 and 1.56 mg
mL . High cell viability was observed for [Lu(ibu) ] and [Y(ibu)
] at concentrations of 6.25; 3.12 and 1.56 mg mL-1
Ibuprofen in association with Gd3+ and Er3+ ions forms [Gd(ibu)
]∙H O and [Er(ibu) ], which display similar
cytotoxicity to pure ibuprofen and are only toxic at 50 mg mL (initial dose). At lower concentrations (6.25, 3.12 and
3
]
-
1
and [Y(ibu)
3 3 3 3
] reduced the cytotoxicity of the drug. At 50 mg mL , [Yb(ibu) ], [Lu(ibu) ] and [Y(ibu)
cytotoxic activity. Analysis of [Yb(ibu)
3
-
1
3
3
3
2
3
-
1
6

-
1
1
.56 mg mL ), [Gd(ibu)
Tb(ibu) ].0.5H O and [Ho(ibu)
.25; 3.12 and 1.56 mg mL . The compounds showed significantly higher values at the initial concentration of 50 mg
]. In addition, [Y(ibu) ] and [Tb(ibu) ].0.5H O also displayed
significantly higher values than ibu at a concentration of 3.12 mg mL . Similarly, at 6.25 mg mL-1, significantly higher
values were observed for [Dy(ibu) ] in relation to ibu. The compounds [Tm(ibu) ], [Tb(ibu) ].0.5H O and [Ho(ibu)
demonstrated significantly lower values compared to ibu at concentration of 25 mg mL , with low values also recorded
3
]∙H
2
O and [Er(ibu)
3 3 3
] also displayed high cell viability. The compounds [Dy(ibu) ], [Tm(ibu) ],
-
1
[
3
2
3
] exhibited cytotoxicity at 50 and 25 mg mL , as well as increased cell viability at
-
1
6
mL-1 when compared to ibu, except for [Dy(ibu)
3
3
3
2
-
1
3
3
3
2
3
]
-
1
-
1
3
for [Ho(ibu) ] at 12.5 mg mL when compared to ibu.
3
.6 Inhibitory concentration of 50% (IC50)
The IC50 concentration is commonly used in experiments aimed at identifying the influence of drugs, extracts and
compounds on different biological systems, and provides the concentration of the inhibitor where the response is
reduced by half. In order to determine these values, the results of the MTT assay were used to perform linear regression,
-
1
producing IC50 values between 22.34 to 47.75 mg mL (Table 4). These were used to carry out biological assays aimed
at evaluating the anti-inflammatory activity of the compounds.
3
.7 Anti-inflammatory activity
Results on the anti-inflammatory activity of the compounds studied summarized in Fig. 6. Inflammatory activity of
the compounds was assessed based on their ability to induce the production of pro-inflammatory metabolites, such as
hydrogen peroxide (H ) and the cytokines TNF-α and IL-10. An inflammatory microenvironment was established as
2
O
2
positive control, induced by human monocytes exposed to lipopolysaccharide (LPS), a molecule present in the cell wall
of Gram-negative bacteria that induces a pro-inflammatory response. In order to assess the immunomodulatory potential
of different compounds, the drugs were added to the LPS-stimulated cultures at a concentration determined by IC50
LPS stimulation confirmed the anti-inflammatory potential of ibuprofen, characterized H , TNF-α and IL-10
production. Similarly, the culture under the effect of [Yb(ibu) ] and [Lu(ibu) ] also exhibited low levels of H and
TNF-α. Despite decreasing TNF-α production, [Y(ibu) ], [Tb(ibu) ]∙0.5H O and [Ho(ibu) ] did not reduce H
production. All the compounds induced a decline in IL-10, except [Yb(ibu) ] and [Lu(ibu) ]. In some cases, ions
associated with ibu result in higher metabolites production when compared to ibu alone. The compounds
Gd(ibu) ]∙H O, [Dy(ibu) ], [Er(ibu) ], [Tm(ibu) ], [Y(ibu) ], [Tb(ibu) ]∙0.5H O and [Ho(ibu) ] increased H
production , while [Gd(ibu) ]∙H O and [Er(ibu) ] induced greater TNF-α production. The compounds [Yb(ibu) ] and
Lu(ibu) ] resulted in higher IL-10 production when compared to ibu.
.
2
O
2
3
3
2 2
O
3
3
2
3
2 2
O
3
3
[
3
2
3
3
3
3
3
2
3
2 2
O
3
2
3
3
[
3
4
. Conclusion
The stoichiometry of the complexes was determined based on thermogravimetric and complexometric titration
results. The TG-DTA curves showed that the compounds presented only loosely bound water, except for gadolinium
and terbium, whose water molecules apparently are in the crystal lattice. Initial mass loss of the anhydrous compounds
7

and comparison of the spectra of gases released indicated propane was the main volatile decomposition product. This is
in agreement with the structural formula of the drug. The energy involved in this reaction was measured by DSC.
Analysis of the IR spectra allowed the identification of two coordination modes for ibu chelating and bridging, or
bridging-chelating. However, since there are no experimental data regarding the geometry around the metal centres, it
was not possible to propose structures for the complexes. In the case of hydrated compounds, the presence of water
molecules may cause bands to overlap, but the split peak is not very evident. X-ray powder diffraction showed that all
the compounds studied exhibited a low degree of crystallinity, with no evidence of isomorphous series. The X-ray
powder diffractometry revealed that all compounds exhibit low degree of crystallinity and no evidence of isomorphous
series.
-
1
Biological tests results showed that ibuprofen was only cytotoxic at a concentration above 50 mg mL , with no
-
1
cytotoxicity observed at 25 mg mL . Ibuprofen induced cell proliferation at the other concentrations tested. With
-
1
respect to compounds tested at higher concentrations (50 and 25 mg mL ), only [Yb(ibu)
reduced the cytotoxic activity of ibuprofen. While [Gd(ibu) ]∙1.0H O and [Er(ibu) ] display similar cytotoxicity to
ibuprofen, positive results were not recorded for [Tb(ibu) ]∙0.5H O, [Dy(ibu) ], [Ho(ibu) ] and [Tm(ibu) ], which
exhibited cytotoxicity at concentrations for which pure ibuprofen was not cytotoxic. However, at lower concentrations
3 3 3
], [Lu(ibu) ] and [Y(ibu) ]
3
2
3
3
2
3
3
3
-
1
(
12.5, 6.25, 3.12, 1.56 mg mL ), all the compounds studied induced cell viability. The results of anti-inflammatory
assessments demonstrated that the [Yb(ibu) ] and [Lu(ibu) ] increased IL-10 production, an anti-inflammatory
mediator, without increasing the production of inflammatory mediators such as TNF-α and H . The other compounds
assessed, [Gd(ibu) ]∙1.0H O, [Dy(ibu) ], [Er(ibu) ], [Tm(ibu) ], [Y(ibu) ], [Tb(ibu) ]∙0.5H O and [Ho(ibu) ], showed no
effective anti-inflammatory potential when compared to ibuprofen, with an increase in H and TNF-α production
3
3
2 2
O
3
2
3
3
3
3
3
2
3
2
O
2
observed in some cases. Taken together, these results suggest that the use of Yb3+ and Lu3+ ions in association with
ibuprofen enhances the anti-inflammatory potential of the drug and reduces its cellular toxicity, making this an effective
option for the formulation of new drugs.
Acknowledgements
The authors wish to thank FAPESP (grant numbers 2012/21450-1; 2013/09022-7 and 2013/04096-2) and CAPES
(
process 024/2012 Pró-equipamentos) for their financial support.
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1
0

Figure Captions
Fig. 1 Structural formula of the acid form of ibuprofen
3 2 3 2 3
Fig. 2 TG-DTA curves of the compounds: (a) [Tb(ibu) ]∙0.5H O, (b) [Ho(ibu) ]∙H O and (c) [Lu(ibu) ]
1
1

3 2
Fig. 3 Infrared spectra of (a) Naibu and (b) [Tb(ibu) ]∙0.5H O
Fig. 4 Spectra of (a) propane from the HR TGA vapor phase Thermo Fisher Scientific Inc. [35] and (b) gas collected at
50ºC of [Tb(ibu) ]∙0.5H O.
2
3
2
1
2

+
3+
3+
3+
3+
Fig 5: Human PBMC were stimulated with compounds of ibuprofen with (a) Yb
(
3
,(b) Lu , (c) Y , (d) Gd , (e) Er ,
f) Dy , (g) Tm , (h) Tb and (i) Ho3+ in different concentrations and evaluated after 96 hours by MTT assay. The
3+
3+
3+
cytotoxicity index is the ratio of test culture (challenged with compounds): culture control (CC). Results are expressed
as mean ± SD, paired t-test, p <0.05. * Represents the difference from CC, represented by the dashed line; # is the
difference between cultures stimulated with ibuprofen and cultures stimulated with the compounds at the same
-
1
concentration (mg mL ).
Fig. 6 Inflammatory mediators. Human monocytes were stimulated with LPS or ibuprofen and mixed for 24 hours.
Inflammatory activity was evaluated by the production of H
2 2
O , IL-10 and TNF-α. Results are expressed as median (25-
7
5 percentile) and minimum-maximum values, Wilcoxon test, p <0.05. * Denotes difference from the culture stimulated
1
3

with LPS only; # Represents differences in cultures stimulated with the compounds in relation to culture stimulated with
only ibuprofen.
Table Captions
Table 1 Analytical data of compounds
Metal / %
TG
Ligand / %
Calc. TG
Water / %
Calc. TG
Final Residue / %
Compound
Calc.
EDTA
20.11
19.90
21.33
20.87
21.02
21.12
21.19
22.07
12.85
Calc.
TG
22.82 23.44
23.75 23.46
23.86 24.79
24.10 23.33
24.33 24.28
Oxide
Gd
Tb
Dy
Ho
Er
[
[
[
[
[
[
[
[
[
3
Gd(ibu) ]∙H
2
O
19.86 20.40
20.20 19.94
20.65 21.59
21.03 20.37
21.28 21.23
21.44 21.37
21.85 21.10
22.04 22.34
12.36 13.08
77.18 76.56
75.10 74.96
75.16 75.21
75.89 76.67
75.67 75.72
75.51 75.60
75.12 75.95
74.93 74.59
82.72 81.80
2.27
1.15
–
1.84
1.58
–
2
O
3
Tb(ibu)
Dy(ibu)
Ho(ibu)
3
]∙0.5H
2
O
4
O
7
3
]
]
2
O
3
3
3
–
–
2
O
Er(ibu)
Tm(ibu)
Yb(ibu)
Lu(ibu)
Y(ibu)
3
]
–
–
2
O
3
3
]
–
–
24.49 24.41 Tm
2
O
3
3
]
–
–
24.88 24.03
25.06 25.41
15.70 16.61
Yb
Lu
2
O
3
3
]
–
–
2 3
O
3
]
–
–
2 3
Y O
Table 2 Temperature ranges of thermal events (), mass losses percentages and peak temperatures observed at each step
of TG-DTA curves of the compounds
1
4

Steps
Third
Compounds
O
First
Second
Fourth
Fifth
[
Gd(ibu)
3
]∙H
2
25-97
159-237
237-531
55.52
–
531-623
623-800

°C
1
.84
7.02
7.92
4.26
m = 6.401 mg
loss/%
–
199 (exo)
542 (exo)
621 (exo)
peak/°C

°C
25-92
1.58
–
152-208
2.46
208-488
57.10
488-506
5.19
506-800
10.21
[
Tb(ibu)
3 2
]∙0.5H O
loss/%
m = 5.305 mg
peak/°C
182 (exo)
452 (exo)
522 (exo)
522 (exo)

°C
–
–
–
151-239
5.59
239-340
7.02
–
340-502
46.91
–
502-800
15.69
[
Dy(ibu)
3
]
loss/%
m = 5.160 mg
peak/°C
206 (exo)
556 (exo)

°C
–
–
–
158-225
4.62
225-370
11.79
–
370-508
44.74
–
508-800
15.52
[
Ho(ibu)
3
]
loss/%
m = 5.225 mg
peak/°C
201 (exo)
545 (exo)

°C
–
–
–
151-223
7.51
223-386
22.77
–
396-509
24.89
–
509-1000
20.55
[
Er(ibu)
3
]
loss/%
m = 5.014 mg
peak/°C
192 (exo)
559 (exo)
[
Tm(ibu)
3
]
°C
–
–
–
160-227
3.69
227-385
15.24
385-515
42.71
515-1000
13.95
loss/%
peak/°C
m = 5.148 mg
203 (exo)
360 (exo)
492 (exo)
551(exo)
2
27-278
2.22
59 (exo)
278-520
45.96
–
520-800
12.40
[
Yb(ibu)
3
]
°C
–
–
–
158-227
5.39
1
m = 5.584 mg
loss/%
peak/°C
3
551 (exo)
194 (exo)
5
28-800
.58
56 (exo)
[
Lu(ibu)
3
]
°C
–
–
–
169-232
2.90
232-373
9.09
–
373-528
53.02
9
m = 5.506 mg
loss/%
peak/°C
5
201 (exo)
507 (exo)
[
Y(ibu)
3
]
°C
loss/%
peak/°C
–
–
–
175-233
4.44
205 (exo)
233-375
9.58
355 (exo)
375-529
58.82
520 (exo)
529-800
10.55
558 (exo)
m = 6.068 mg
1
5

Table 3 Spectroscopic data for ibuprofen sodium salt complex and the lanthanide and yttrium (III)

a(COO) / cm-1
1587

s(COO) / cm-1
 / cm-1
Compound
Naibu
1405
182
[
Gd(ibu)
3
]∙H
2
O
1543
1411, 1427
1414, 1427
1412, 1427
1412, 1427
1413, 1427
1414, 1428
1413, 1429
1414, 1429
1412, 1427
132, 116
135, 122
129, 114
132, 117
130, 116
130, 116
128, 112
128, 113
131, 116
[
Tb(ibu) ]∙0.5H
3
2
O
1549
[
[
Dy(ibu)
3
]
]
1541
Ho(ibu)
3
1544
[
Er(ibu)
Tm(ibu)
Yb(ibu)
Lu(ibu)
Y(ibu)
and
3
]
1543
[
3
]
1544
[
3
]
1541
[
3
]
1542
[
3
]
1543

a(COO)

s(COO)
=
anti-symmetrical and symmetrical vibrations of the COO group, respectively;

= [a(COO)] – [s(COO)]
Table 4 IC50 values of PBMC cultures stimulated with [Gd(ibu)
Er(ibu) ], [Tm(ibu) ], [Yb(ibu) ], [Lu(ibu) ] and [Y(ibu) ].
3 2 3 2 3 3
]∙H O, [Tb(ibu) ]∙0.5H O, [Dy(ibu) ], [Ho(ibu) ],
[
3
3
3
3
3
-
1
Compound
IC50 (mg mL )
33.65
Hibu
[
Gd(ibu)
3
]∙H
2
O
30.40
[
Tb(ibu) ]∙0.5H
3
2
O
27.19
[
[
Dy(ibu)
3
]
]
31.46
Ho(ibu)
3
30.56
[
Er(ibu)
Tm(ibu)
Yb(ibu)
Lu(ibu)
Y(ibu)
3
]
36.57
[
3
]
29.58
[
3
]
47.75
[
3
]
37.53
[
3
]
22.34
1
6