Pt(II) and Pd(II) complexes with ibuprofen hydrazide: Characterization, theoretical calculations, antibacterial and antitumor assays and studies of interaction with CT-DNA

Article abstract of DOI:10.1016/j.molstruc.2017.10.072

Palladium(II) and platinum(II) complexes with a hydrazide derivative of ibuprofen (named HIB) were synthesized and characterized by chemical and spectroscopic methods. Elemental and thermogravimetric analyses, as well as ESI-QTOF-MS studies for both compl

Full text of DOI:10.1016/j.molstruc.2017.10.072

Accepted Manuscript
Pt(II) and Pd(II) complexes with ibuprofen hydrazide: Characterization, theoretical
calculations, antibacterial and antitumor assays and studies of interaction with CT-
DNA
Carlos M. Manzano, Fernando R.G. Bergamini, Wilton R. Lustri, Ana Lúcia T.G. Ruiz,
Ellen C.S. de Oliveira, Marcos A. Ribeiro, André L.B. Formiga, Pedro P. Corbi
PII:
S0022-2860(17)31418-7
10.1016/j.molstruc.2017.10.072
MOLSTR 24443
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 17 August 2017
Revised Date: 18 October 2017
Accepted Date: 19 October 2017
Please cite this article as: C.M. Manzano, F.R.G. Bergamini, W.R. Lustri, Ana.Lú.T.G. Ruiz, E.C.S.
de Oliveira, M.A. Ribeiro, André.L.B. Formiga, P.P. Corbi, Pt(II) and Pd(II) complexes with ibuprofen
hydrazide: Characterization, theoretical calculations, antibacterial and antitumor assays and studies of
interaction with CT-DNA, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.10.072.
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ACCEPTED MANUSCRIPT
Graphical Abstract – Manuscript Corbi et al.
Crystal structure of the Pd(II) complex with ibuprofen hydrazide. CT-DNA interaction studies by
circular dichroism and SYBR Green fluorescence.

1
ACCEPTED MANUSCRIPT
Pt(II) and Pd(II) complexes with ibuprofen hydrazide: characterization, theoretical
calculations, antibacterial and antitumor assays and studies of interaction with CT-
DNA
a
a
b
Carlos M. Manzano , Fernando R. G. Bergamini , Wilton R. Lustri , Ana Lúcia T. G.
c,d
c
e
a
a*
Ruiz , Ellen C. S. de Oliveira , Marcos A. Ribeiro , André L. B. Formiga , Pedro P. Corbi
a
Inorganic Chemistry Department, Institute of Chemistry, University of Campinas –
UNICAMP, P.O. Box 6154, 13083-970, Campinas, SP, Brazil.
b
Biological and Health Sciences Department, University of Araraquara - UNIARA, 14801-
3
20 Araraquara, SP, Brazil
c
Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA),
University of Campinas – UNICAMP, 13148-218, Paulínia, SP, Brazil.
d
Faculty of Pharmaceutical Sciences, University of Campinas - UNICAMP, 13083-871,
Campinas, SP, Brazil.
e
Chemistry Department, Federal University of Espírito Santo – UFES, 29075-910, Vitória-
ES, Brazil
*
Corresponding author:
Institute of Chemistry – University of Campinas.
P.O. Box 6154, 13083-970, Campinas, SP, Brazil
Telephone: + 5519 35213130 / FAX: + 5519 35213023
E-mail address: ppcorbi@iqm.unicamp.br

2
ACCEPTED MANUSCRIPT
Abstract
Palladium(II) and platinum(II) complexes with a hydrazide derivative of ibuprofen
named HIB) were synthesized and characterized by chemical and spectroscopic methods.
(
Elemental and thermogravimetric analyses, as well as ESI-QTOF-MS studies for both
-
complexes, confirmed a 1:2:2 metal/HIB/Cl molar ratio. The crystal structure of the
palladium(II) complex was solved by single crystal X-ray diffractometric analysis, which
permitted identifying the coordination formula [PdCl (HIB) ]. Crystallographic studies also
2
2
indicate coordination of HIB to the metal by the NH group. Nuclear magnetic resonance
2
and infrared spectroscopies reinforced the coordination observed in the crystal structure
and suggested that the platinum(II) complex presents similar coordination modes and
structure when compared with the Pd(II) complex. The complexes had their structures
optimized with the aid of DFT methods. In vitro antiproliferative assays showed that the
[
PdCl (HIB) ] complex is active over ovarian cancer cell line OVCAR-03, while biophysical
2 2
studies indicated its capacity to interact with CT-DNA. The complexes were inactive over
Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa bacterial strains.
Keywords: Platinum(II), Palladium(II), Ibuprofen hydrazide, Spectroscopic analysis,
DFT studies, Antiproliferative assays.

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Abbreviation List
CD - Circular dichroism
CSD - Crystal Structure Database
CT-DNA - Calf-thymus deoxyribonucleic acid.
E. coli - Escherichia coli Gram-negative bacterial strain.
ESI-QTOF-MS - Electrospray ionization time of flight mass spectrometry
HIB - ibuprofen hydrazide
ibu - ibuprofen
IR - infrared vibrational spectroscopy
Non-tumor human cell line - HaCat (immortal keratinocyte).
NSAID - non-steroidal anti-inflammatory drugs
P. aeruginosa - Pseudomonas aeruginosa Gram-negative bacterial strain.
[PdCl (HIB) ] or PdHIB - Palladium complex with ibuprofen hydrazide
2 2
[
PtCl (HIB) ] or PtHIB - Platinum complex with ibuprofen hydrazide
2
2
S. aureus - Staphylococcus aureus Gram-positive bacterial strain.
SSNMR - Solid state nuclear magnetic resonance
SYBR Green or SG – Nucleic acid electrophoresis stain: N',N'-dimethyl-N-[4-[(E)-(3-
methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-
propylpropane-1,3-diamine.
Tumor human cell lines - U251 (glioma); UACC-62 (melanoma); MCF-7 (breast); NCI-
ADR/RES (multidrug resistant ovarian); NCI-H460 (lung, non-small cells); PC-3 (prostate);
OVCAR-03 (ovarian); HT-29 (colon); K562 (leukemia);

4
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Introduction
The discovery of the capacity of cisplatin to inhibit cellular division in Escherichia coli
by Barnett Rosenberg et al. in 1960´s and the further report about its action over tumor cells
can be considered the starting point of a new era on synthesis and development of metal-
based drugs in medicine [1]. Nowadays, cisplatin remains as one of the most used drugs
against different types of cancer, including lung, head and neck, testis, and ovarian tumors
[
2,3]. Although cisplatin and their derivatives (e.g. carboplatin and oxaliplatin) have been
extensively used with good results in cancer treatment, several limitations can be
highlighted, such as the increasing of cisplatin cross-resistance in tumors and its
nephrotoxicity and neurotoxicity. The effective delivery of cisplatin also appears to be a
challenge to be overcome. The interactions of cisplatin and derivatives with other
biomolecules in the organism can lead to adverse effects [4,5] and reduce the amount of
the compound that achieve its target. High doses of the platinum-based drugs, which are
needed to reach an effective intracellular concentration and perform their activities can lead
to severe systemic toxicity [6].
Although a great effort has been made over the past decades to treat cancer more
safely and effectively, an increasing rate of cancer incidence and mortality is observed. So,
the search for new drugs for cancer treatment is of great importance. One of the strategies
in drug discovery relies on the association of the cytotoxic properties of a certain metal ion
to the potential synergistic activity offered by a specific ligand. An interesting approach is
the use of commercial drugs with favorable safety profile, known pharmacokinetics and
adequate permeability as ligand [7].
In special, many types of cancer are related to inflammation responses [8] and recent
studies showed that the non-steroidal anti-inflammatory drugs (NSAIDS) were able reduce
cancer risk development of tumors such as colon, breast, lung, and prostate [9]. Ibuprofen

5
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-1
(
ibu, C H O , M.W. 206.29 g·mol , Figure 1A) is one of the most common NSAIDs used
13 18 2
in the clinics for the treatment of headache, muscular ache, arthritis rheumatoid, fever and
pain related to tissue inflammation [10].
Concerning treatment and prevention of post-surgical infections, NSAIDs are
commonly used concomitantly to antibiotics to reduce the level of tissue inflammation [11]
.
Therefore, anti-inflammatory ligands are particularly interesting for association with metal
ions in the design and development of new antimicrobial or anticancer agents.
Hydrazides and their derivatives have shown to have a broad pharmacological action,
including antimicrobial and antitumor activities. For hydrazides and hydrazones of NSAIDs,
the modification of the acidic site of these molecules are desired partly reduce the adverse
effects to the gastrointestinal tract [12,13]. There are several examples of biological active
hydrazides [14], some of them are isonicotinohydrazide as an antituberculosis drug [15] and
N'-isopropylisonicotinohydrazide with antidepressant activity [16]. Sulfonyl hydrazide
derivatives exhibit a wide range of bacteriostatic activity and can act as DNA modifying
agents. Some of them exhibited antitumor activities against murine tumors, including the
B16 melanoma, M109 lung carcinoma, L1210 leukemia, P388 and M5076 reticulum cell
sarcoma [17].
There are examples of metal complexes with ibuprofen that exhibits antibacterial
activities such as the silver(I) and gold(I) ones [18,19], which are active against Gram-
-
positive and Gram-negative bacterial strains. Also, a platinum(II) ibuprofenate (ibu )
complex was shown to be active against Gram-positive microorganisms [20]. Nevertheless,
no metal complexes with ibuprofen hydrazide seems to be reported so far.
This work presents the synthesis, characterization and biological studies of new
palladium(II) and platinum(II) complexes with a hydrazide (Figure 1B) derived from
ibuprofen. The crystal structure of the palladium(II) complex was determined by single-

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crystal X-ray diffraction studies. Antiproliferative and antimicrobial assays of both
complexes were performed and studies of their interactions with calf thymus DNA (CT-
DNA) are also described.
Figure 1. Schematic structure of (A) ibuprofen and (B) ibuprofen hydrazide with numbered
carbon atoms. The hydrogen atoms were assigned with the same index as the carbon
atoms.
Experimental section
Materials and Methods
DNA sodium salt from calf-thymus type I (CT-DNA), SYBR Green I (10,000x in
DMSO) and K PtCl were purchased from Sigma–Aldrich Laboratories. Ibuprofen (racemic
2
4
mixture) was from Maxpharm. K PdCl and hydrazine hydrate (64%) were purchased from
2
4
Acros Laboratories. Methyl chloroformate (99%), dichloromethane (99%) and triethylamine
99%) were from Spectrum Chemical Mfg, LabSynth and Merck Laboratories, respectively.
Triethylamine was purified by distillation with potassium carbonate while dichloromethane
(

7
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was purified by distillation with phosphorous pentoxide (P O ) prior to the syntheses. Methyl
2
5
chloroformate was also purified by distillation prior to the syntheses. Elemental analyses
were performed on a Perkin Elmer 2400 CHNS/O Analyzer. Electronic absorption spectra in
the range 190–1100 nm were acquired using a 1.0 cm quartz cuvette in a diode array
HP8453 UV/Vis absorption spectrophotometer. Infrared spectroscopy measurements (IR)
were performed on an Agilent Cary 630 FTIR spectrometer, using the attenuated total
-1
reflectance (ATR) method. The IR spectra were recorded from 400-4000 cm , with
-1
1
13
resolution of 2 cm . The H and C nuclear magnetic resonance (NMR) spectra of HIB,
[
PdCl (HIB) ] and [PtCl (HIB) ] were recorded in a Bruker Avance III 500 MHz (11.7 T)
2
2
2
2
15
1
13
1
spectrometer. The { N, H} and { C, H} HSQC and HMBC NMR spectra were recorded in
a Bruker Avance III 400 MHz (9.4 T) spectrometer. Samples were analyzed at 298K. The
6
[
PdCl (HIB) ] complex was analyzed in a DMSO-d solution, while the [PtCl (HIB) ] was
2
2
2
2
3
analyzed in methanol-d due to its better solubility and stability in this solvent. HIB was
3
6
analyzed in both methanol-d and DMSO-d . The chemical shifts for all solution
13
15
measurements were given relative to tetramethylsilane (TMS). The C and N solid state
NMR (SSNMR) spectra were recorded in a Bruker 300 MHz spectrometer, using the
combination of cross-polarization, proton decoupling and magic angle spinning techniques
(CP/MAS). Electrospray ionization mass spectrometry (ESI-MS) measurements were
carried out using a Waters Quattro Micro API. Samples were evaluated in the positive
mode. Each solution was directly infused into the instrument’s ESI source and analyzed in
the positive mode, with capillary potential of 3.00 kV, trap potential of 2 kV, source
temperature of 150 °C and nitrogen gas for desolvati on.
Single-crystal X-ray diffraction
X-Ray diffraction data for the [PdCl (HIB) ] complex were collected on a Bruker APEX
2
2

8
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II Duo diffractometer using Mo-Kα radiation (λ = 0.71073 Å) from a fine-focus sealed tube
with a curved graphite monochromator. The data collection, cell refinement and data
reduction were performed by CrysAlisPro 1.171.38.46 (Rigaku OD, 2015). The isopropyl
group C9B, C10B, C11B and C12B, Figure 2, was found to be disordered, with refined
occupations of 0.345 and 0.655.
All non-hydrogen atoms were refined anisotropically. The hydrogen atoms in the
compounds were added to the structure in idealized positions and further refined as riders
[
21]
OLEX2 [22]. Illustrations were rendered using the Pov-Ray tool on Mercury CSD 3.8
Copyright CCDC 2001-2016) [http://www.ccdc.cam.ac.uk/mercury/].
. The crystal data are presented on Supplementary Information #1. Molecular graphics:
(
Geometry optimizations
The theoretical calculations of the [PdCl (HIB) ] and [PtCl (HIB) ] complexes were
2
2
2
2
carried out using density functional theory (DFT) with a PBE0 hybrid functional [23] and the
def2-TZVP basis set [24] for all atoms using ORCA software [25]. The geometric
-5
-
optimization was performed with a convergence criterion of 10 a.u. for the gradient and 10
8
a.u. for the density. The RIJCOSX method [26] was used for the calculation of the
exchange energy, using the standard number of points for the grid calculations. The
structures were confirmed as the minimum of the potential energy surface (PES) using the
same level of theory. Illustrations were rendered using the Pov-Ray tool on Mercury CSD
3
.8 (Copyright CCDC 2001-2016) [http://www.ccdc.cam.ac.uk/mercury/].
Synthesis of ibuprofen hydrazide (HIB)
The synthesis of HIB was performed similarly to the methodology described in the
-3
literature by Nakka et al. [27]. Ibuprofen (2.0x10 mol) was dissolved in 20 mL of dry

9
ACCEPTED MANUSCRIPT
dichloromethane followed by the addition of 300 ꢀL of trimethylamine. The solution was
stirred for 5 minutes at room temperature and then transferred to an ice bath (0-5 °C ).
Methyl chloroformate (200 ꢀL) was added to the cold solution and the mixture was
kept under stirring for 30 min. The solution was then slowly transferred to a flask containing
5
00 ꢀL of hydrazine hydrate (N H ·6H O, 60%) and then maintained under stirring, at first
2 4 2
in reflux (~50 °C , 3 hours) and then for additional 18 hours without heating. Finally, the
reaction was quenched with 20 mL of distilled water and the volume of the resulting
suspension was reduced under low pressure evaporation. The solid obtained was collected
by filtration, washed with cold water and dried under P O .
4
10
Anal. Calc. for C H N O (%): C, 70.87; H, 9.15; N, 12.72. Found (%): C, 70.99; H,
13
20
2
-1
9
.24; N, 12.74. The yield of the synthesis was 71.0%. IR (cm ): 3271 (νNH), 2960-2850
+
+ 1
(
νCH), 1687 (νCO), 1638 (δNH ). ESI-QTOF-MS (m/z) 221.1649 [C H N O+H ] . H NMR
2 13 20 2
6
(
500 MHz, DMSO-d ) δ 9.13 (s, 1H), 7.21 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H), 4.17
(s, 1H), 3.47 (q, J = 7.0 Hz, 1H), 2.39 (d, J = 7.1 Hz, 2H), 1.91 – 1.64 (m, 1H), 1.31 (d, J =
1
3
6
7
1
7
.1 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H). C NMR (125 MHz, DMSO-d ) δ 173.39, 139.80,
1
39.72, 129.17, 127.46, 44.72, 43.40, 30.09, 22.65, 18.84. H NMR (500 MHz, CD OD) δ
3
.27 (d, J = 8.1 Hz, 2H), 7.11 (d, J = 8.1 Hz, 2H), 3.58 (q, J = 7.1 Hz, 1H), 2.46 (d, J = 7.2
13
Hz, 2H), 1.98 – 1.75 (m, 1H), 1.46 (d, J = 7.1 Hz, 3H), 0.91 (d, J = 6.6 Hz, 6H). C NMR
(
125 MHz, CD OD) δ 175.09, 140.15, 138.59, 128.84, 126.75, 47.61, 44.62, 43.86, 30.06,
3
13
15
2
1.28, 17.27. C SSNMR: δ 179.6, 140.9, 129.9, 46.6, 43.6, 31.7, 24.8, 17.3. N SSNMR:
δ 134.4/127.2, 56.3.
Synthesis of [PdCl (HIB) ] and [PtCl (HIB) ] complexes (PdHIB and PtHIB)
2
2
2
2
For the synthesis of the complexes, potassium tetrachloropalladate(II) or potassium
-4
tetrachloplatinate(II) (2.5x10 mol) were first dissolved in a minimum volume of distilled

1
0
ACCEPTED MANUSCRIPT
-4
water. Then, HIB (5.0x10 mol) was dissolved in 5 mL of methanol and slowly added to the
solution of the metal salts. Immediately, yellow solids precipitated. The reaction was
maintained under stirring for 2 hours for the palladium(II) complex and 20 hours for the
platinum(II) one.
The obtained compounds were collected by filtration, washed with methanol and
distilled water and dried under P O . Single crystals of the [PdCl (HIB) ] complex suitable
4
10
2
2
for X-ray diffraction studies were obtained by slow evaporation of an ethanolic solution of
the complex. Anal. Calc. for PdC H N O Cl complex (%): C, 50.53; H, 6.52; N, 9.07.
26
40
4
2
2
-1
Found (%): C, 50.27; H, 6.48; N, 9.13. Yield: 84.7%. IR (cm ): 3239 (νNH), 2960-2850
+
+
(
νCH), 1668 (νCO). ESI-QTOF-MS (m/z) 545.2001 [Pd(C H N O) -H ] , 583.1688
13 20 2 2
+
1
6
[
Pd(C H N O) Cl] . H NMR (500 MHz, DMSO-d ) δ 7.21 (dd, J = 8.0, 2.8 Hz, 2H), 7.07
13 20 2 2
(d, J = 8.0 Hz, 2H), 3.69 (q, J = 6.9 Hz, 1H), 3.50 (q, J = 6.9 Hz, 1H), 2.39 (d, J = 7.1 Hz,
13
2
H), 1.91 – 1.65 (m, 1H), 1.31 (d, J = 7.1 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H). C NMR (125
MHz, DMSO) δ 173.40/172.08, 140.01, 138.63, 129.28, 127.66, 44.72, 43.33/42.65, 30.08,
1
3
2
1
2.65, 18.90. C SSNMR: δ 175.0, 139.9, 138.3, 128.6, 45.95/44.18, 31.90/31.03, 23.76,
15
9.90. N SSNMR: δ 139.55/136.03/131.06, 18.13/11.75.
Anal. Calc. for PtC H N O Cl (%): C, 44.19; H, 5.71; N, 7.93. Found (%): C, 45.39;
26
40
4
2
2
-1
H, 5.53; N, 6.97. Yield: 65.8%. IR (cm ): 3251 (νNH), 2960-2850 (νCH), 1666 (νCO). ESI-
+
+
1
QTOF-MS (m/z) 634.2774 [Pt(C H N O) -H] , 671.2528 [Pt(C H N O) Cl] . H NMR
13
20
2
2
13 20
2
2
(
500 MHz, CD OD) δ 7.27 (2H), 7.17 – 6.93 (2H), 3.63 (1H), 2.47 (2H), 1.95 – 1.72 (1H),
3
1
3
1
4
2
.47 (3H), 0.91 (6H). C NMR (125 MHz, CD OD) δ 140.51, 137.48, 129.01, 126.98,
3
13
4.64, 43.58, 30.05, 21.31, 17.27. C SSNMR: δ 173.47, 139.58, 129.19, 45.12, 30.37,
15
2.70, 18.17. N SSNMR: δ 138.09, 9.66.
Antibacterial assays in vitro

1
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ACCEPTED MANUSCRIPT
In this study, Gram-positive Staphylococcus aureus (ATCC 25923), and Gram-
negative Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853)
bacterial strains were inoculated in tubes containing BHI (Brain Heart Infusion KASVI) and
incubated for 18 hours at 35-37 °C . Appropriate amou nts of each bacterial suspension were
added to new tubes in BHI sterile culture medium until the 1.0 nephelometric McFarland
8
-1
turbidity scale (~3.0x10 CFU·mL ; CFU = colony forming units). Samples (HIB,
-1
[
PdCl (HIB) ] and [PtCl (HIB) ]) suspended in DMSO (15 µg·µL ) were added (50 µL/well)
2 2 2 2
to the second well of the 96 well microplate, followed by the addition of sterile BHI (50
µL/well). In the next 6 wells, a serial dilution (half) in BHI sterile were performed,
maintaining the final volume of 100 µL per well. After that, the bacterial suspensions were
added (100 µL/well) to each serial dilution reaching 0.5 in the McFarland turbidimetric scale
8
-1
-
(
~1.5x10 CFU·mL ) and the sample final concentrations ranged from 58.6 to 3750 µg·mL
1
.
The first well of the plate was used as bacterial growth control (untreated bacteria
8
-1
suspension, ~1.5x10 CFU·mL ). The microplates were incubated for 18h at 35-37°C in a
wet chamber under stirring (100 rpm). After the incubation time, sterile aqueous solution of
resazurin (0.02%, 15 ꢀL/well) were added. The absorbance measurements were performed
after further incubation for 3 hours [28].
Antiproliferative assays in vitro
The antiproliferative activities of HIB, [PdCl (HIB) ] and [PtCl (HIB) ] were investigated
2
2
2
2
over nine human cancer cell lines (U251 = glioma, UACC-62 = melanoma; MCF-7 = breast;
NCI-ADR/RES = multidrug resistant ovarian; NCI-H460 = lung, non-small cells; PC-3 =
prostate; OVCAR-03 = ovarian; HT-29 = colon; K562 = leukemia). The tumor cell lines were
provided by Frederick Cancer Research & Development Center, National Cancer Institute,
Frederick, MA, USA. The cell proliferation was also evaluated using a non-tumor cell line

1
2
ACCEPTED MANUSCRIPT
HaCat (human keratinocyte), provided by Dr. Ricardo Della Coletta (University of
Campinas- UNICAMP, Brazil). The same panel of cells were used previously in other
assays performed in our group [29].
Stock cultures were grown in 5 mL of RPMI-1640 supplemented with 5% fetal bovine
®
serum (RPMI/FBS 5% Gibco , USA) at 37 °C in 5% CO with a 1% penicillin:streptomycin
2
®
-1
mixture (Nutricell , Brazil, 1000 U·mL ) (complete medium) [30,31]. The samples HIB,
[
PdCl (HIB) ], [PtCl (HIB) ], K [PdCl ] and K [PtCl ] were prepared directly in complete
2
2
2
2
2
4
2
4
-1
medium (5 mg·mL ) and then diluted in the same medium, affording the final
-1
concentrations of 0.25, 2.5, 25 and 250 ꢀg·mL . Doxorubicin (final concentrations of 0.025,
-1
0
.25, 2.5 and 25 ꢀg·mL in complete medium) was used as positive control.
4
-1
Cells in 96-well plates (100 ꢀL/well, inoculation density: 3.5 to 6 x 10 cell·mL ) were
exposed to different concentrations of sample and controls (100 ꢀL/well) in triplicate, for 48
h at 37 °C and 5% of CO . Before (T plate) and after the sample addition (T plates), the
2
0
1
cells were fixed with 50% trichloroacetic acid (50 ꢀL/well), and the cell proliferation was
determined by the spectrophotometric quantification (540 nm) of the cellular protein content
using the sulforhodamine B assay [32]. The GI values (concentration that inhibits 50% of
50
cell growth) were determined through sigmoidal regression using Origin 8.0 software
OriginLab Corporation, USA).
(
Biophysical studies: interaction with DNA
In this assay, CT-DNA was incubated for 24h at 37 °C (100 µmol·L CT-DNA
-1
-1
aqueous solution in 10 mmol·L NaClO , 1% DMSO), with HIB, [PdCl (HIB) ] and
4
2
2
-1
[
PtCl (HIB) ] in ratios [compound]/[DNA] of 0.1, 0.2 and 0.3 (10, 20 and 30 µmol·L ). The
2
2
solutions were used for circular dichroism spectroscopy experiments and for the
®
fluorescence experiments with SYBR Green I . Fluorescence measurements were

1
3
ACCEPTED MANUSCRIPT
performed on a Cary Eclipse fluorescence spectrophotometer. Excitation and emission slits
were 5 nm and scanning speed was set to medium. A quartz cuvette of four clear windows
®
and optical path length of 1.0 cm was used. SYBR Green I (SG) was used as fluorescence
probe. Excitation wavelength was set as 485 nm and emission window from 505 to 700 nm.
For each sample the fluorescence at 520 nm (maximum for SG) was monitored with the
consecutive addition of SG (4µL of SG 5x). Circular Dichroism measurements were
performed in a Jasco J-720 spectrophotometer, from 205 to 320 nm, with a scanning speed
-1
of 20 nm·min and 4 accumulations. The Quartz cuvettes of 1.0 cm of optical path length
were used.
Results and discussion
Single crystal X-ray diffraction
Crystals of the [PdCl (HIB) ] complex were obtained by recrystallization in ethanol and
2
2
its structure was determined by single crystal X-ray diffraction. The crystallographic data
are summarized in Table 1. Additional crystallographic data are presented in
Supplementary material #1, Table S1.
The complex crystallized in the P2 /c space group and the asymmetric part of the unit
1
cell is presented in Figure 2(A). As one may observe, the asymmetric unit contains one
Pd(II) ion, two chloride ions and two neutral HIB molecules. The complex possess a near
perfect square geometry with the least square plane defined by those 4 ligands atoms with
r.m.s of 0.0290. The Pd1 ion is deviate 0.0154 Å from the plane defined from N1B, N1A,
Cl1 and Cl2. The hydrazide groups of the HIB molecules coordinated trans to each other
(N1A-Pd-N1B = 178.58(12) °) . In addition, two chlori de ions complete the sphere
coordination (Cl1-Pd-Cl2 = 177.41(3) °) . For similar Pd(II) and Pt(II) compounds reported in
the literature [33–38] the trans isomers were predicted by DFT calculations to be the most

1
4
ACCEPTED MANUSCRIPT
stable geometry, with the exception of the Pt(II) complexes with 4-methoxybenzylcarbazate,
benzyl carbazate and 3-methoxybenzoic acid hydrazide [39], in which the cis isomers are
suggested. This feature indicates that other factors such as steric hindrance, ligand basicity
as well as intermolecular interactions may be responsible for the additional stabilization of
such system in comparison to cis-coordinated complexes.
Table 1. Crystallography data for [PdCl (HIB) ].
2
2
Empirical formula
C H Cl N O Pd
26 40 2 4 2
-1
Formula weight (g·mol )
617.92
150
T (K)
γ(Mo Kα) (Å)
Crystal system
Space group
a (Å)
0.71073
Monoclinic
P2 /c
1
27.8148 (5)
8.9319 (3)
11.5187 (3)
92.462 (2)
2859.05 (13)
4
b (Å)
c (Å)
β
3
V (Å )
Z
ꢀ
3
D (Mg·m )
1.436
x
-
ꢀ
(mm ¹)
0.865
1280
F(000)
Number Par./ Ind. Refl./ Rint
361/ 5855/ 0.037
0.043; 0.103; 1.05
1.92 and -1.19
2
2
2
R[F > 2σ(F )]; wR(F ); S
-3
∆
〉max and ∆〉min (e Å )

1
5
ACCEPTED MANUSCRIPT
Figure 2. (A) View of asymmetric part of unit cell of the complex [PdCl (HIB) ]. (B)
2
2
Representation of the hydrogen bonds observed among the moieties [symmetry codes:(i) 1-
x,-y,1-z, (ii) x, 1/2-y, -1/2+z, (iii) 1-x, 1-y, 1-z]. In a seek of clarity, the disorder on the
isopropyl group was intentionally not represented. Probability level of ellipsoids: 50%.
The bonds distances and angles from both HIB molecules are in good agreement with
those observed for ibuprofen and ibuprofenate found on CSD (Tables S2 and S3 -
Supplementary Material #1). An important feature observed in the crystal structure is the
presence of both R and S configurations of HIB molecule (related to C2 chiral center)
coordinated to Pd(II) ion. Since the ligand was synthesized from a racemic mixture of
ibuprofen and no further racemic purification was performed, it is reasonable to suggest that
both configurations would be present in the complex. Moreover, the bond lengths, angles
and torsions among Pd(II) and its first sphere of coordination is close to those reported in
the literature for a similar structure [40]. Such values are presented in Supplementary
Material #1, Tables S4 and S5.

1
6
ACCEPTED MANUSCRIPT
The presence of chlorine ions contribute to the packing of crystal structure since Cl1
i
…
ii
acts as receptor in two hydrogen bonds with N1b (i=1-x,1-y,1-z) (D A) (3.365(4)) and N1b
…
…
(
ii=x,1/2-y,-1/2+z) ((D A 3.484(4)) atom. The interaction with H1bb ((A H 3.365(4)) is
forming a dimeric structure with another complex moiety while the interaction with H1ba
atom forms a chain in the c direction. Besides it, the Cl2 ion appears as acceptor in other
iii
…
iv
…
two hydrogen bonds formed with N1A (D A 3.197(3) iii=x,1/2-y,1/2+z) and N2b (D A
3
.157(4) iv=1-x,-y,1-z). Finally, the presence of the chlorine ions in the structure play an
important role in the stabilization of supramolecular structure.
Infrared spectroscopy
The IR spectra of the compounds are shown in Figure 3. In the spectrum of the ligand
-1
the absorption corresponding to N-H bond is observed at 3271 cm , while in the spectrum
-1
-1
of the complexes the same bands are shifted to 3239 cm and 3251 cm for PdHIB and
-1
PtHIB, respectively. The region between 2960 and 2850 cm , which presents vibrational
bands due to the C-H bond is almost identical in all spectra. It was expected since the
coordination does not occur on the neighborhood of the C-H bonds [41,42].
-1
-1
The C=O absorption is observed at 1687cm in the ligand and it is shifted to 1668cm
-1
in the PdHIB spectrum and to 1666 cm for PtHIB. The NH deformation appear at 1638
2
-1
cm in the spectrum of the ligand, while for the complexes this absorption band is no longer
observed due to the coordination [42]. This behavior is consistent with the coordination by
the NH group as it was already showed by crystal structure of PdHIB.
2

1
7
ACCEPTED MANUSCRIPT
Figure 3. Infrared spectra of HIB (A), [PdCl (HIB) ] (B) and [PtCl (HIB) ] (C).
2
2
2
2
NMR spectroscopic measurements
The characterization of the ligand and complexes was performed by solution NMR,
1
13
13
1
13
1
including H, C, { C- H} HSQC (heteronuclear single quantum coherence), { C- H}
15
1
15
1
HMBC (heteronuclear multiple bond coherence), { N- H} HSQC and { N- H} HMBC NMR.
6
4
The [PdCl (HIB) ] and [PtCl (HIB) ] complexes were analyzed in DMSO-d and methanol-d
2
2
2
2
solutions, respectively. The choice of methanol for the study of the [PtCl (HIB) ] complex
2
2
was due to the stability of the complex in this solvent. The stability studies by UV-vis
spectroscopy are presented in Supplementary material #2, Figures S4 and S5. Therefore,
for comparison purposes, HIB was analyzed in both solvents. All solution state NMR
spectra can be found in Supplementary material #2, Figures S6-S18.
In order to evaluate coordination of HIB to Pd(II) and Pt(II) and guarantee chemical
shifts representative of the bulk, without the possible interference of coordinating solvents,

1
8
ACCEPTED MANUSCRIPT
13
15
15
C and N NMR experiments were also performed in solid state. In the case of N NMR
experiments in special, those were performed only in solid state also due to solubility level
1
15
limitations and the further impossibility of obtainment of { H, N} HSQC spectra of the
complexes in solution. Furthermore, as a comparative, the ligand HIB was also evaluated
by the same methods.
13
The C SSNMR spectra of the ligand and the respective complexes are presented in
15
Supplementary material #2, Figures S19-S21. The N SSNMR of the same compounds are
13
15
shown in Figure 4. Table 2 summarizes the chemical shifts observed for both C and N
NMR experiments of the ligand and the respective complexes.
15
Figure 4. N SSNMR spectra of HIB (A), [PdCl (HIB) ] (B) and [PtCl (HIB) ] (C).
2
2
2
2
Multiple peaks in the PdHIB and HIB spectra are observed probably due to the
presence of polymorphism. The main difference in chemical shifts between the spectrum of
HIB and the spectra of the complexes is in the nitrogen of the group NH , which is shifted
2

1
9
ACCEPTED MANUSCRIPT
from 56.3 ppm for HIB to 11.7/18.1 ppm for [PdCl (HIB) ] and 9.7 ppm for [PtCl (HIB) ].
2
2
2
2
This is an evidence that coordination to Pd(II) and Pt(II) occurs by the NH of the hydrazide
2
group and does not involve the NH moiety.
These data are also consistent with the IR experiments, suggesting the same
13
coordination site. Also, no significant chemical shifts were observed in the C SSNMR
spectra of PdHIB and PtHIB indicating that no coordination occurs by the carboxylic groups.
13
15
Table 2. C and N SSNMR chemical shifts for HIB, [PdCl (HIB) ] and [PtCl (HIB) ].
2
2
2
2
Chemical shifts (ppm)
Carbon numbers
Nitrogen atoms
13
11/12 10
2
9
4 /5/6 /7
3
8
1
NH
NH2
1
27.2
HIB 17.3 24.8 31.7 43.6 46.6 129.9 140.9 140.9 179.6
56.3
1
1
34.4
31.1
31.0
1.9
11.7
18.1
PdHIB 19.9 23.8
44.2 45.0 128.6 138.3 139.9 175.0
136.0
39.5
3
1
PtHIB 18.2 22.7 30.4 45.1 45.1 129.2 139.6 139.6 173.5
138.1
9.7
Mass spectrometric analysis
+
The mass spectra of the [PdCl (HIB) ] complex (Figure 5) shows the ion [Pd(HIB) Cl]
2
2
2
at m/z 583.1874 (error 32.6 ppm) as the most abundant peak. It also shows the presence of
+
the ion [Pd(HIB) -H] at m/z 545.2149, which refers to the complex with one of the ligands
2
bearing a negative charge due to deprotonation. For [PtCl (HIB) ] complex (Figure 6) the
2
2
+
results are similar, with the ions [Pt(HIB) Cl] at m/z 671.2528 (error 9.2 ppm) and [Pt(HIB)
2
2
+
–
H] at m/z 634.2934.

2
0
ACCEPTED MANUSCRIPT
Figure 5. (A) Mass spectrum for the [PdCl (HIB) ] complex from 100 to 1000 m/z. (B)
2
2
+
Theoretical isotope pattern for [Pd(HIB) Cl] at m/z 583.1874 and (C) experimental isotopic
2
pattern. The notation HIB refers to C H N O.
13
20
2
Figure 6. (A) Mass spectrum for the [PtCl (HIB) ] complex from 100 to 1000 m/z. (B)
2
2
+
Theoretical isotope pattern for [Pt(HIB) Cl] of m/z 617.2485. (C) Experimental isotopic
2
pattern. The notation HIB refers to C H N O.
13
20
2

2
1
ACCEPTED MANUSCRIPT
DFT calculations
The geometric calculations for [PdCl (HIB) ] and [PtCl (HIB) ] complexes were
2
2
2
2
optimized using density functional theory (DFT). The DFT data, including the atomic
coordinates for the optimized structures, are presented in Supplementary Material #1,
Tables S6-S7.
As previously observed, the crystal structure of the Pd(II) complex is composed of
one Pd(II) coordinated by two neutral HIB molecules and two chloride ions. The elemental
and analysis, as well as the ESI-QTOF-MS of the Pd(II) complex in its powder form,
indicate that the same composition is observed in the crystal and powder samples. In
addition, the same studies regarding the Pt(II) complex indicates that it also possesses a
1
:2:2 metal/HIB/chloride ratio, as observed for the Pd(II) complex. Moreover, the SSNMR
and IR spectroscopic measurements of the powder samples indicate that, for both
complexes, the coordination occurs by the hydrazide moieties of HIB. Therefore, it is
reasonable to assume that the Pt(II) complex is coordinated by HIB in the same fashion as
observed in the [PdCl (HIB) ] crystal structure.
2
2
The [PdCl (HIB) ] DFT optimized structure is presented in Supplementary Material #1
2
2
(
Figure S2), while the [PtCl (HIB) ] optimized structure is presented in Figure 7.
2 2
Figure 7. PBE0/def2-TZVP optimized structure of [PtCl (HIB) ]. The atomic coordinates
2
2
obtained using the DFT are presented in Supplementary Material #1, Table S7.

2
2
ACCEPTED MANUSCRIPT
Both optimized structures of Pd(II) and Pt(II) complexes possess HIB ligands with
bond lengths and angles similar to those observed in the crystal structure of the Pd(II)
complex (see Table S8 - Supplementary Material #1). Furthermore, as one may observe in
Table 3, the bond distances for Pd(II) and the ligand atoms are very close to the values
observed in the crystal structure.
In the case of Pt(II) complex, on the other hand, the same selected bonds were
compared to the crystal structure of a similar complex previously reported in the literature
[
43]. Again, the bond lengths for the Pt(II) complex are very close to those observed in the
literature. In a different way of the optimized structure of the Pt(II) complex, in which the HIB
molecules are arranged in a similar fashion [PdCl (HIB) ] crystal structure, the HIB ligands
2
2
on DFT optimized structure of the Pd(II) complex are arranged at different positions (see
Figure S3 - Supplementary Material #1). The planes formed among the carbon atoms of the
benzyl ring of the HIB molecules in the crystal structure are observed displaced by 80.15°
and 30.07°of their respective planes in the DFT op timized structure. Since intermolecular
interactions are observed in the crystal structure that would not be otherwise simulated by
the DFT model (which considers a single molecule in vacuum), it is reasonable to assume
that the most thermodynamically stable structure obtained by DFT can be different to that
observed in the crystal.
The DFT optimized structures of [PdCl (HIB) ] and [PtCl (HIB) ] were compared with
2
2
2
2
other DFT optimized structures found in the literature for Pd(II) and Pt(II) complexes that
have NH moieties coordinated in trans geometry (Table 3). It is interesting to notice that in
2
these complexes the Pd-N bond lengths calculated using the PBE0 hybrid functional are
very similar to each other and to the distances reported in the crystal structures. On the
other hand, the same lengths calculated using the B3LYP functional are generally longer,

2
3
ACCEPTED MANUSCRIPT
suggesting that the functional PBE0 better predicts this kind of coordination bond.
Table 3. Selected bond lengths for the Pd(II) and Pt(II) complexes in comparison to the
literature and to the crystal structure of [PdCl (HIB) ]. The bond lengths calculated by DFT
2
2
are presented in average.
M-Cl2/
M-Cl1*
.2889(9)/ 2.048(3)/
M-N1B/
M-N1A
N1B-N2B/ C1B-O1B/
N1A-N2A
1.408(4)/
1.425(4)
C1A-O1A
1.226(5)/
1.224(4)
2
[
PdCl (HIB) ] crystal
2 2
2
.309(1)
2.036(3)
‡
‡
‡
‡
[
PdCl (HIB) ] DFT
2.307
2.046
1.406
1.214
2
2
‡
‡
[
PdCl (C H N ) ] [34]
2.337
2.049
-
-
2
10 12 2 2
‡
‡
[
PdCl (L) ]** [37]
2.34
2.07
-
-
-
-
-
-
-
-
2
2
†
[
[
[
Pd(C H NO S) ] [36]
Pd(C H N O ) ] [33]
Pd(C H NO S) ] [35]
-
-
-
2.089
6
10
3
2
†
2.08
2.11
11
11
2
2 2
†
6
10
2
2
2
2
.296(2)/
2.051(5)/
2.028(6)
1.416(8)/
1.419(9)
1.227(5)/
1.210(9)
§
Pt(II) complex crystal
.299(2)
‡
‡
‡
‡
[
PtCl (HIB) ] DFT
2.308
2.049
1.407
1.215
2
2
†
[
Pt(C H N O ) ] [38]
-
2.08
-
-
11
11
2
2 2
*
M refers to the dicationic metal ion, M(II) (i.e. Pd(II) or Pt(II)).
** L refers to carbazates and hydrazides (mean value).
§
Z. Kristallogr. 209 (1994) 293. [43]
calculated using the PBE0 functional
calculated using the B3LYP functional
‡
†
Minimal inhibitory concentration (MIC)
Neither [PdCl (HIB) ] nor [PtCl (HIB) ] complexes inhibited any bacterial growth in the
2
2
2
2
experimental conditions. Nonetheless, the ligand HIB was able to inhibit S. aureus (MIC =
-1
-1
-1
-1
1
850 µg·mL , 8.4 mmol·L ) and E. coli (MIC = 3750 µg·mL , 17.0 mmol·L ). The
antibacterial capacities of HIB were not described in the literature to date. Although HIB
was able to inhibit bacterial growth, it could not be considered as a promisor antibacterial
agent since a stringent endpoint criteria at MIC < 25 µM was proposed for anti-infectious
pure compounds [44].

2
4
ACCEPTED MANUSCRIPT
Antiproliferative assays
The antiproliferative activities of HIB as well as [PdCl (HIB) ] and [PtCl (HIB) ]
2
2
2
2
complexes were obtained against a human tumor panel. Doxorubicin, a frontline drug used
for the treatment of several types of cancer [45], was used as positive control and the
precursor salts, K [PdCl ] and K [PtCl ], were also evaluated. This activity was expressed
2
4
2
4
as the parameter GI50 representing the sample concentration necessary to inhibit 50% of
cell growth, therefore a low GI50 means a high antiproliferative activity. The results are
-1
reported by estimated concentration (µmol·L ) (Table 4).
-1
The [PtCl (HIB) ] complex presented GI values higher than 353.2 µmol·L for all the
2
2
50
cell lines. Besides, the [PdCl (HIB) ] complex showed selective inhibition against the
2
2
-1
ovarian cancer cell line (OVCAR-03, GI50 = 27.0 ꢀmol·L ) (Table 4). Another important
aspect of antiproliferative activity is the selectivity towards cancer and normal cells.
Experimentally, this selectivity can be estimated using a selectivity index (SI), defined as
[
46]:
ꢃꢁ50ꢂ(ꢄꢅꢆꢅꢇ)
ꢀ
ꢁ =ꢂ
ꢃꢁꢈꢉꢂ(ꢊꢅꢋꢊꢌꢍꢂꢊꢌꢎꢎꢂꢎꢏꢋꢌ)
Where GI50 (HaCat) is the GI50 for the non-tumor human cell line (immortalized
keratinocytes) and GI50 (cancer cell line) is the GI50 value for a given cancer cell line.
Hypothetically, the higher the value of SI the higher the dose that could be administered
safely [46]. The values of SI calculated for each cell line are presented in Table 4.
Antiproliferative profiles are presented in the Supplementary Material #3, Figures S22 -
S27.

2
5
ACCEPTED MANUSCRIPT
-1
Table 4. GI50 (ꢀmol·L ) and selectivity index (SI) values obtained for HIB, [PdCl (HIB) ],
2
2
[
PtCl (HIB) ], K [PdCl ], K [PtCl ] and doxorubicin in the in vitro antiproliferative assay.
2
2
2
4
2
4
-1
GI (µmol·L )
5
0
UACC-
NCI-
NCI-
OVCAR
-03
U251
MCF-7
PC-3
HT-29 K562 HaCat
62
ADR/RES H460
HIB
511.10 144.34 171.12
*
*
*
*
*
521.54 92.14
*
*
*
119.83 217.42
PdHIB
PtHIB
*
*
*
*
*
*
*
*
27.02
*
*
*
>404.6
353.24
*
K PdCl
99.25 10.72 96.50
64.08 4.09 61.91
90.68
20.23
0.46
117.94 96.80
57.81 20.71
<0.045 0.42
11.33
23.37
0.092
96.80 85.16 82.71
66.49 59.02 6.99
0.26 0.055 <0.045
2
4
K PtCl
2
4
Doxorubicin <0.045 <0.045 <0.045
Selectivity Index (SI)
UACC-
NCI-
NCI-
OVCAR
-03
U251
MCF-7
PC-3
HT-29 K562
62
ADR/RES H460
HIB
PdHIB
PtHIB
K PdCl
0.43
1.51
1.27
*
*
*
0.42
*
2.36
14.97
*
*
*
1.81
*
*
*
*
*
*
*
*
*
*
*
*
*
0.83
0.11
*
7.71
1.71
*
0.86
0.11
*
0.91
0.35
<0.09
0.70
0.12
*
0.85
0.34
<0.11
7.30
0.30
<0.49
0.85
0.11
0.97
0.12
2
4
K PtCl
2
4
Doxorubicin
<0.17 <0.81
GI : concentration that inhibits 50% cell growth or cytostatic effect after 48h-exposition. SI: selectivity index
50
calculated as GI50 (HaCat)/ GI50 (cancer cell line). Human tumor cell lines: U251 = glioma, UACC-62 = melanoma;
MCF-7 = breast; NCI-ADR/RES = multidrug resistant ovarian; NCI-H460 = lung, non-small cells; PC-3 =
prostate; OVCAR-03 = ovarian; HT-29 = colon; K562 = leukemia; Human non-tumor cell line: HaCat =
immortalized keratinocytes
Considering the ovarian cancer cell line (OVCAR-03), the [PdCl (HIB) ] complex has
2
2

2
6
ACCEPTED MANUSCRIPT
shown a SI equal to 14.97, which is higher than the SI for doxorubicin (SI ꢁ 0.49), K PdCl
2
4
(
SI = 7.30) and K PtCl (SI = 0.30). Although the activity of [PdCl (HIB) ] was lower than
2 4 2 2
doxorubicin, its selectivity towards cancer cells was noticeable. The activity observed
against OVCAR-3 is particularly interesting once this cell line is used as a model for
cisplatin resistance in ovarian cancer [47,48]. In addition, HIB showed low activity against
the cell lines tested, but its SI was higher than 1.0 for the cancer cell lines UACC-62
(melanoma), MCF-7 (breast), OVCAR-03 (ovarian) and K562 (leukemia).
Biophysical studies: interaction of the compounds with DNA
The interactions between bio-macromolecules and drugs have attracted special
interest during the past few decades [49]. Given that DNA is an important target for cisplatin
and many metal complexes, the interaction between them have been studied extensively
[
50–54]. The damage caused by anticancer agents in the DNA of cancer cells block their
division and lead to cell death [55]. The interaction with DNA may occur in many ways,
including covalent binding and non-covalent modes such as intercalation, groove binding
and electrostatic interaction. To investigate the presence of these interactions, numerous
techniques are available, including UV spectroscopy, circular dichroism and fluorescence
studies with DNA intercalators [56–60]. In the present work, fluorescence studies and
circular dichroism spectroscopic measurements were performed.
Fluorescence studies with SYBR Green (SG)
SYBR Green is a specific fluorophore that effectively interacts with double strand
DNA, more specifically in the DNA minor groove [61], increasing its fluorescence emission
upon complex formation by >1,000-fold [62]. Therefore, it is possible to study the interaction
of a selected compound with DNA by observing the influence of the sample on the

2
7
ACCEPTED MANUSCRIPT
fluorescence emission intensity of the interaction SG-DNA.
In this study, CT-DNA was incubated for 24h at 37 °C with HIB, PdHIB and PtHIB in
the ratios (compound/DNA) of 0.1, 0.2 and 0.3. For each sample, the fluorescence at 520
nm (SG maximum) was monitored before and after consecutive addition of 4 ꢀL of SG (SG
5
x) until 40 ꢀL (SG final volume). The fluorescence of SG alone was measured and it is
insignificant, thus not reported. For the samples of DNA incubated with HIB and
PtCl (HIB) ] no significant fluorescence suppression was observed for all the
[
2
2
concentrations tested.
In samples of DNA incubated with [PdCl (HIB) ] (Figure 8), at the ratios of 0.2 and 0.3,
2
2
the fluorescence intensity was decreased significantly suggesting an interaction with DNA.
These results suggested an intercalation of [PdCl (HIB) ] between the DNA bases or
2
2
interaction with the DNA groove.
Figure 8. Relative fluorescence intensities of SG at 250 nm for samples of free DNA and
DNA in the presence of [PdCl (HIB) ] in the ratios (compound/DNA) 0.1, 0.2 and 0.3.
2
2
Circular dichroism spectroscopy (CD)
Circular dichroism (CD) is widely used to monitor different conformational forms of
DNA, among many other applications [59,60]. It is possible to differentiate DNA

2
8
ACCEPTED MANUSCRIPT
conformational changes among B-form, A-form and Z-form as well as other structures due
to interactions with different compounds. The CT-DNA is characteristically a B-form right-
handed DNA as observed in the CD spectrum (Figure 9), where there is a positive band at
2
75 nm due to base stacking and a negative band at 245 nm attributed to helicity [59,63]. In
addition, a positive band at 220 nm was also observed [60,64]. The obtained results
showed that HIB and [PtCl (HIB) ] did not induce any noticeable changes in the CD
2
2
spectrum even in the highest ratio tested (data not shown).
As expected from fluorescence studies, the [PdCl (HIB) ] complex promoted
2
2
alterations in the DNA conformation (Figure 9). At ratio (RI) 0.1 (compound/DNA), it was
observed a decrease in the intensity of all the bands and a slightly bathochromic shift, but
the spectrum was still characteristic of a B-form DNA. Further at RI= 0.2 and RI=0.3, the
CT-DNA suffered a significant destabilization of the B-form. The band intensities decreased
and there was a considerable shift in the band at 275 nm suggesting stacking alteration
probably due to intercalation or covalent binding. Furthermore, the changes in the band at
2
45 nm indicated that the complex modified the DNA helicity [50].
Figure 9. CD spectra of CT-DNA in absence and presence of [PdCl (HIB) ] in the ratios
2
2