UNIVERSIDADE ESTADUAL DE CAMPINAS

INSTITUTO DE QUÍMICA

AMANDA DE ALMEIDA E SILVA

SYNTHESIS, CHARACTERIZATION AND EVALUATION OF THE

ANTIBACTERIAL AND ANTIPROLIFERATIVE ACTIVITIES OVER TUMOR
CELLS OF Pt(II) AND Cu(II) COMPLEXES WITH NAPROXEN AND
NAPROXEN HYDRAZIDE

SÍNTESE, CARACTERIZAÇÃO E AVALIAÇÃO DA ATIVIDADE

ANTIBACTERIANA E ANTIPROLIFERATIVA SOBRE CÉLULAS TUMORAIS
DE COMPLEXOS DE Pt(II) E Cu(II) COM NAPROXENO E HIDRAZIDA DO
NAPROXENO

CAMPINAS
2023
 AMANDA DE ALMEIDA E SILVA

SYNTHESIS, CHARACTERIZATION AND EVALUATION OF THE

ANTIBACTERIAL AND ANTIPROLIFERATIVE ACTIVITIES OVER TUMOR
CELLS OF Pt(II) AND Cu(II) COMPLEXES WITH NAPROXEN AND
NAPROXEN HYDRAZIDE

SÍNTESE, CARACTERIZAÇÃO E AVALIAÇÃO DA ATIVIDADE

ANTIBACTERIANA E ANTIPROLIFERATIVA SOBRE CÉLULAS TUMORAIS
DE COMPLEXOS DE Pt(II) E Cu(II) COM NAPROXENO E HIDRAZIDA DO
NAPROXENO

Dissertação de Mestrado apresentada ao Instituto de

Química da Universidade Estadual de Campinas como
parte dos requisitos exigidos para a obtenção do título de
Mestra em Química na área de Química Inorgânica.

Master's Dissertation presented to the Institute of

Chemistry of the University of Campinas as part of the
requirements to obtain the title of Master in Chemistry in
the area of Inorganic Chemistry.

Supervisor: Dr. Pedro Paulo Corbi

Escolher um item.

O arquivo digital corresponde à versão final da Dissertação defendida pela

aluna Amanda de Almeida e Silva e orientada pelo Prof. Dr. Pedro Paulo
Corbi.

CAMPINAS
2023
 Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca do Instituto de Química
Simone Luiz Alves - CRB 8/9094

Silva, Amanda de Almeida e, 1993-

Si38s SilSynthesis, characterization and evaluation of the antibacterial and
antiproliferative activities over tumor cells of Pt(II) and Cu(II) complexes with
naproxen and naproxen hydrazide / Amanda de Almeida e Silva. – Campinas,
SP : [s.n.], 2023.

SilOrientador: Pedro Paulo Corbi.

SilDissertação (mestrado) – Universidade Estadual de Campinas, Instituto de
Química.

Sil1. Platina. 2. Cobre. 3. Anticâncer. I. Corbi, Pedro Paulo, 1974-. II.

Universidade Estadual de Campinas. Instituto de Química. III. Título.

Informações Complementares

Título em outro idioma: Síntese, caracterização e avaliação da atividade antibacteriana e

antiproliferativa sobre células tumorais de complexos de Pt(II) e Cu(II) com naproxeno e
hidrazida do naproxeno
Palavras-chave em inglês:
Platinum
Copper
Anticancer
Área de concentração: Química Inorgânica
Titulação: Mestra em Química na área de Química Inorgânica
Banca examinadora:
Pedro Paulo Corbi [Orientador]
Wilton Rogério Lustri
Wdeson Pereira Barros
Data de defesa: 25-05-2023
Programa de Pós-Graduação: Química

Identificação e informações acadêmicas do(a) aluno(a)

- ORCID do autor: https://orcid.org/0000-0002-4312-9395
- Currículo Lattes do autor: http://lattes.cnpq.br/4435093500727966

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BANCA EXAMINADORA

Prof. Dr. Pedro Paulo Corbi (Orientador)

Prof. Dr. Wilton Rogério Lustri (UNIARA)

Prof. Dr. Wdeson Pereira Barros (UNICAMP)

A Ata da defesa assinada pelos membros da Comissão Examinadora, consta no

SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa da Unidade.

Este exemplar corresponde à redação

final da Dissertação de Mestrado
defendida pela aluna Amanda de
Almeida e Silva, aprovada pela
Comissão Julgadora em 25 de maio de
2023.
This work is dedicated to…

My grandmother Benedicta Sampaio e Silva,

that completed her 97 years.

My parents Leila Maciel de Almeida e Silva

and Osmar de Souza e Silva Jr for

their support along my trajectory.

All that supported me so that I could achieve my aims,

and that will always support me.

This work also represents my tribute to everyone who, unfortunately, because of SARS-CoV-2,
lost their lives. The pandemic affected my master's degree, and also affected me directly... Still,
I was immensely enlightened and my family was protected from the terrible evils that this
disease brought to our world. Thank you, my God, for always protecting me, as well as my
family and friends!
“I was taught that the way of progress was neither swift nor easy.”

Marie Curie

“Não se espante com a altura do voo. Quanto mais alto, mais longe do perigo. Quanto
mais você se eleva, mais tempo há de reconhecer uma pane. É quando se está próximo
do solo que se deve desconfiar.”

Santos Dumont

“Tudo vale a pena quando a alma não é pequena.”

Fernando Pessoa

“A carreira científica é fascinante”

Prof. Dr. Oswaldo Luiz Alves

 Aknowledgments

This work was carried out with the support of the Coordination for the Improvement of Higher
Education Personnel – Brazil (CAPES) – Financing Code 001, thank you for the master's
scholarship granted.

This work also counted with the support of (2018/12062-4; 2021/08717-8) Fundação de
Amparo à Pesquisa do Estado de São Paulo – FAPESP.

To the support of FAEPEX (2432/22) for financing the XX BMIC congress and my lasts
months in Campinas with the “auxílio-ponte”, we give our thanks.

I thank CNPq (407012/2018-4) for the financial support to our research group.

I also would like to sincerely thank...

My advisor Pedro Paulo Corbi, for his immense and most holy patience, for always being so
understanding and positive in the face of all the tribulations at work and in life. To “senhor
Chefe” my eternal gratitude for all support.

The partner professors of this work, Prof. Wilton Rogério Lustri for his affection and
willingness, Prof. Ana Lúcia for her dedication, Prof. Ana Maria for the expertise and Prof.
Douglas Pereira for his readiness and to all students who participated directly in this work.

The IQ-Unicamp technicians, in particular: Claudinha, Anderson, Diego, Carol, Fabi, Ricardo
and Millene for their support not only in the analyzes per se, but also in terms of knowledge
and support.

The professors Wdeson and Camilla, who contributed to this work in Qualify.

The professors Wilton Rogério and Wdeson, once more, for the suggestions during my final
presentation.

My professors at PED, Prof. Sigoli, Prof. Wdeson and Prof. Corbi, my PED-mates and to my
dearest PED students who encouraged me to pursue an academic career and ensured that I was
on the right path.

My professors in the Master's disciplines, Oswaldo Luiz Alves (in memorium) who was very
dear to everyone and who I miss a lot, Jackson Dirceu (IQ) who gave me a lot of support in the
presentations, Matheus Borba (CNPEM) who taught me how to draw in Inkscape and make my
figures and slides prettier, Denize Favaro and Cláudio Tormena (IQ) for the knowledge
aqcuired in NMR.

My friends from LQBM Gabi, Laura, Diego, Chicão and Letícia, from lab I 103 Marcelo,
Maurício, Silmar Marcus, Acácia, Mariana, Débora and Giovanna and from lab I 113 Mateus,
Luís Gustavo, Mariana, Fiorella, João P, Gabriel, Nycolas and Evandro for their immeasurable
support both at work and in life and for being a united group that gave me so many happy days.

The former LEMB – UFMG, where I had my base in laboratory chemistry and learned a lot
from the girls, especially Ana Paula and Ana Délia, and from my advisors, Prof. Rubén, Prof.
Esperanza and Prof. Luciano.

The former IMM research group for accepting me and teaching me so much about synthesis
and microscopy. Special thanks to Prof. Steven Bell, who gave me the courage to change from
chemical engineering to pure chemistry. A super big thanks to Taifur, who teached me much
more than just chemistry, and is my friend for life.

The former POPLE – UFS, where I started my scientific career, in computational chemistry.
Special thanks to my supervisor Prof. Viviane Felicíssimo.

To my friends that UNICAMP brought to my life Bruno, Taís, Neuryelen, Grazi, Jéssica, Igor,
Toninho and Laiane, who always help me with what they can and root for me.

The friends I made at UFS, Queen’s University Belfast and UFMG, for supporting me in my
academic career. A special thanks for Yessiquita, Fabrício, Richard, Renato, Jordania, Vanessa,
Mirlene, Pedro, Fábio da Nasa, Cabeludo do Deutsch, Aulus, Umberto, Keyla,,and Audi.

My friends that life gave me: John, Misticos, Drik, Maximo Guerreiro, Flatudonos, Rex,
Ramiro, Andréa and Aninha, Aline, Vânia, Idaci, Anuzia, Bel, Renata, Hudson, Xande, Sorriso,
Vascaíno, Paulo Cabeludo, Fábia, Sandrinha, among many others that I keep in my heart.

My childhood friends, my bro Luiz Fernando, my sis Dani, and the CSL gang, for their
everlasting friendship and affection. You live forever in my heart.

Ieda and the professionals from Cecom - Unicamp, in special: Fábio, Ricardão, Guilherme,
Ricardo and Cláudia, who made many moments of this journey lighter and healthier. Your
support has been invaluable! I also would like to thank the other professionals who
accompanied me during this period, in special Dr. Roberto and Dra. Fernanda.
My warrior cousin Lavínia, and to my cousin Lilian and my uncle Roosevelt for their support
at various stages of my life.

My amazing aunts Liane and Marly, who always rooted for me with great faith and affection in
their hearts.

My great aunts (in memorium) Benvinda and Neuza, whose priceless affection keeps my heart
company.

My grandparents (in memorium) Léa, who made me seize every opportunity with both hands,
José Augusto, my model of resilience, and Osmar whose affection transcends the distance
between Heaven and Earth.

My grandmother Benê, who prays every day that I succeed in what I do and always encourages
me to move forward.

The other loved ones in my family, who may not know details of where I am in this world, but
live in my heart.

My parents Leila and Osmar, the inspiration of my life, who never doubted that I would make
it this far and always supported me with the greatest love in the world, my eternal and loving
Thank you!

São Judas Tadeu and Angel Gabriel, to whom I am eternally devoted, for never leaving my side
and taking care of everyone who lives in my heart. To all Saints who accompany my family.
To Our Mother, who with her maternal warmth brought peace to my heart in the most difficult
moments. To Jesus, who inspires us all.

And above all, my thanks to God for the gift of life, for the graces achieved and for leading my
trajectory, in which I lacked nothing.

Muitíssimo obrigada!!! Thanks so much!!!!!

 ABSTRACT

This work brings a new perspective to the studies on the use of naproxen (Nap) and naproxen
hydrazide-based complexes of copper(II) and platinum(II) as antiproliferative agents. The
platinum complex with naproxenate (Pt-Nap) was identified as the square planar cis-
[PtNap2(DMSO)2] isomer. The copper complex with naproxenate (Cu-Nap) presented itself in
a binuclear paddle-wheel structure respecting a proportion of 1 Cu: 2 Nap : 1 H2O. Both
complexes were inactive against the Gram-positive and Gram-negative strains tested. Pt-Nap
presented low cytostatic behavior over tumor cells, but a good viability for normal cells, while
Cu-Nap was cytotoxic against all cells and even slightly cytocidal against glioma tumoral cells
at 150 μg/mL (138.9 μmol/L). Naproxen hydrazide (Hyd) was successfully synthesized and
identified in ATR-FTIR, (15N, 1H) peaks in 2D NMR and 15N SSNMR. The Pt(II) complex with
naproxen hydrazide (Pt-Hyd) presented a square planar trans structure, with the Pt(II) metal
center coordinated to the terminal NH2 from hydrazide, without proton loss. Both Hyd and Pt-
Hyd did not present antibacterial effect. Hyd presented a low cytocidal effect against 786-0
(renal) and U251 (glioma) tumor cells and was highly cytostatic against NCI/ADR-RES
(ovarian multidrug resistant) and HT-29 (colon) at 150 μg/mL (64.68 μmol/L). Pt-Hyd was
cytostatic against all tumor cells tested and highly cytocidal against renal and glioma tumoral
cells at 15 μg/mL (19.41 μmol/L). However, Pt-Hyd was highly cytostatic over 3T3 (mice
fibroblast) and highly cytocidal over HaCaT (human keratinocyte) normal cells at 15 μg/mL
(19.41 μmol/L). Hyd was also cytocidal against HaCaT, but not cytostatic to 3T3, in the highest
concentration tested of 150 μg/mL (64.68 μmol/L). The obtained results confirm the potential
of application of the naproxen-based complexes over tumor cells and further studies are
envisaged to attest such biological activities.
 RESUMO

Este trabalho traz uma nova perspectiva para os estudos sobre o uso de complexos de cobre(II)
e platina(II) associados ao naproxeno (Nap) e hidrazida do naproxeno como agentes
antiproliferativos. O complexo de platina com naproxenato (Pt-Nap) foi identificado como um
isômero cis-[PtNap2(DMSO)2] quadrado planar. O complexo de cobre com naproxenato (Cu-
Nap) apresentou-se em uma estrutura dinuclear tipo paddle-wheel respeitando a proporção de
1 Cu: 2 Nap : 1 H2O. Ambos os complexos foram inativos contra as cepas Gram-positivas e
Gram-negativas testadas. Pt-Nap apresentou baixo comportamento citostático sobre células
tumorais, mas boa viabilidade para células normais, enquanto Cu-Nap foi citotóxico contra
todas as células e até ligeiramente citocida contra células tumorais de glioma a 150 μg/mL
(138.9 μmol/L). A hidrazida de naproxeno (Hyd) foi sintetizada com sucesso e identificada no
ATR-FTIR, nos picos de (15N, 1H) em 2D NMR e no 15
N SSNMR. O complexo Pt(II) com
hidrazida do naproxeno (Pt-Hyd) apresentou uma estrutura trans quadrado-planar, com o centro
metálico Pt(II) coordenado ao NH2 terminal da hidrazida, sem perda de prótons. Tanto Hyd
quanto Pt-Hyd não apresentaram efeito antibacteriano. Hyd apresentou baixo efeito citocida
contra células tumorais 786-0 (renal) e U251 (glioma), e foi altamente citostático contra
NCI/ADR-RES (ovariano multirresistente) e HT-29 (cólon) na concentração de 150 μg/mL
(64.68 μmol/L). Pt-Hyd foi pelo menos altamente citostático contra todas as células tumorais
testadas e altamente citocida contra células tumorais renais e de glioma na concentração de 15
μg/mL (19.41 μmol/L). No entanto, Pt-Hyd foi altamente citostático sobre 3T3 (fibroblastos de
camundongos) e altamente citocida sob células normais HaCaT (queratinócitos humanos) a 15
μg/mL (19.41 μmol/L). Hyd também foi citocida contra HaCaT, mas não citostático para 3T3,
mesmo na concentração mais alta testada de 150 μg/mL (64.68 μmol/L). Os resultados obtidos
confirmam o potencial de aplicação dos complexos à base de naproxeno sobre células tumorais
e estudos futuros são necessários para atestar tais atividades biológicas.
 LIST OF FIGURES

Figure 1. Mechanisms of action of transition metal complexes over tumor 23

eucaryotic cells. Adapted from Marlova et al [17].

Figure 2. Mechanism of action of metal complexes over bacteria. Adapted from 24

Mikhailova[18] and Bogdan et al[19].

Figure 3. Schematic representation of (a) naproxen; (b) naproxen hydrazide. 27

Figure 4. Schematic representation of the hydrazone formation. 28

Figure 5. Numbered structure of NaNap. 33

Figure 6. Scheme of the experimental procedure of Hyd synthesis. 35

Figure 7. Numbered scheme of the molecular form of Hyd. 36

Figure 8. Scheme of Disc Diffusion method. 38

Figure 9. Scheme of a panel of tumoral cells assay. 40

Figure 10. Q-Orbitrap ESI-MS (positive mode, 1%DMSO, 200μL/min in 44

MeOH:H2O, dilution 20% MeOH:H2O, no column, 3.5kV, R=3.5x104) spectrum
of Hyd: (a) Full spectrum; (b) zoom on experimental spectrum; (c) corresponding
simulated spectrum.

Figure 11. ATR-FTIR of Hyd (blue) in comparison with Nap (orange) ranging 45
from: (a) 4000 to 500 cm-1; (b) 1800 to 500 cm-1(zoom).

Figure 12. 1H and 13C NMR (500 MHz, D2O) of NaNap. 46

Figure 13. 1H and 13C NMR (500 MHz, DMSO-d6) of Hyd. 47

Figure 14. 15N SSNMR (10 kHz) of Pt-Hyd. 48

Figure 15. Q-Orbitrap ESI-MS (positive mode, 1%DMSO, 200μL/min in 50

MeOH:H2O, dilution 20% MeOH:H2O, no column, 3.5kV, R=3.5x104) spectra of:
(a) Cu-Nap (no HCOOH) full spectrum; (b) zoom on Cu-Nap experimental
spectrum; (c) simulated spectrum for Cu-Nap; (d) Pt-Nap (0.1% HCOOH) full
spectrum; (e) zoom on Pt-Nap experimental spectrum; (f) simulated spectrum for
Pt-Nap.

Figure 16. Q-Orbitrap ESI-MS (negative mode, 15 μL/min in MeOH:H2O, 50x 51

dilution 20% MeOH:H2O, 1%DMSO, no column, 3.5kV, R=3.5x104) spectra of
Cu-Nap: (a) Full spectrum; (b) zoom on Cu-Nap experimental spectrum; (c)
simulated spectrum for Cu-Nap.

Figure 17. TG/DTA from 20° to 800°C with increase rate of 10°C per minute of: 52
(a) Cu-Nap; (b) Pt-Nap.

Figure 18. ATR-FTIR from (a) 4000-1200 cm-1 of Cu-Nap, Pt-Nap and NaNap; 54
(b) 1300 to 500 cm-1 of Pt-Nap and NaNap; (c) 500 to 100 cm-1 of Cu-Nap and Pt-
Nap.

Figure 19. Raman spectra of the complexes Cu-Nap and Pt-Nap, and their starting 55
materials NaNap and cis-[PtCl2DMSO2].

Figure 20. Kinetic UV-Vis spectra of Cu-Nap from 0 to 24h in DMSO. 57

Figure 21. (a) Structural formula for the Cu-Nap complex; (b) Molecular model 59
for Cu-Nap.

Figure 22. (a) 1H NMR (500 MHz, DMSO-d6) of Pt-Nap; (b) 13C NMR (500 MHz, 61
DMSO-d6) of Pt-Nap.

Figure 23. HSQC {13C, 1H} (500 MHz, DMSO-d6) of Pt-Nap. 62

Figure 24. HMBC {13C, 1H} (500 MHz, DMSO-d6) of Pt-Nap. 63

195
Figure 25. Pt NMR (500 MHz, DMSO-d6) spectra for Pt-Nap (top) and cis- 64
[PtCl2(DMSO)2] (bottom).

Figure 26. Kinetic 1H NMR(500 MHz, DMSO-d6) spectrum of Pt-Nap in t = 0h, 65

0.5h, 1h, 2h, 4h, 6h, 12h and 24h.
Figure 27. Molecular models of Pt-Nap isomers: (a) trans [Pt(Nap)2(DMSO)2]; (b) 66
cis- [Pt(Nap)2(DMSO)2].
Figure 28. Q-Orbitrap ESI-MS ((positive mode, 1%DMSO, 200 μL/min in
67
MeOH:H2O, dilution 20% MeOH:H2O, no column, 3.5kV, R=3.5x104) of Pt-Hyd:
(a) Full spectrum; (b) zoom of Pt-Hyd experimental spectrum; (c) simulated
spectrum for Pt-Hyd.

Figure 29. TG/DTA of Pt-Hyd, from 20° to 900°C with increase rate of 10°C per 69
minute.

Figure 30. ATR-FTIR of Hyd (blue) and Pt-Hyd (beige) from (a) 4000-500 cm-1; 70
(b) 1800-500 cm-1 (zoom).

Figure 31. 1H NMR (500 MHz, DMSO-d6) of Pt-Hyd (bottom) comparatively to 71

Hyd (top).

Figure 32. 13C NMR (500 MHz, DMSO-d6) of Pt-Hyd (bottom) in comparison to 72
Hyd (top). The vertical lines in Pt-Hyd spectrum refer to the signals that indicate
mixture of products, likely isomers.

Figure 33. HMBC {13C, 1H} (500 MHz, DMSO-d6) of Pt-Hyd. 73

Figure 34. 2D NMR (500 MHz, DMSO-d6) HSQC {15N, 1H} of Pt-Hyd. 74

Figure 35. 15N SSNMR (10 kHz) of Pt-Hyd. 74

Figure 36. Molecular models of Pt-Hyd isomers: (a) trans-[Pt(Hyd)2Cl2]; (b) cis- 77
[Pt(Hyd)2Cl2].

Figure 37. Disc diffusion of Pt-Nap, Cu-Nap, NaNap, cis-[PtCl2(DMSO)2] over: 79

A) S. aureus, B) B. cereus, C) E. coli, D) P. aeruginosa.

Figure 38. Antiproliferative profile (%) for data in μg/mL of (a) cis- 82
PtCl2(DMSO)2; (b) NapNa; (c) CuCl22H2O; (d) Pt-Nap; (e) Cu-Nap

Figure 39. Antiproliferative profile (%) of (a) K2PtCl4; (b) Hyd; (c) Pt-Hyd. 83
Figure 40. HaCaT cytotoxicity for the synthesized compounds on this work, in 84
comparison to NaNap.

Figure A1. HSQC{15N,1H} NMR (500 MHz, DMSO-d6) of Hyd. 101

Figure A2. HMBC {15N,1H} NMR (500 MHz, DMSO-d6) of Hyd 102

Figure A3. EPR results for Cu-Nap in the solid state at: (a) Room temperature; (b) 103
77K.
 LIST OF TABLES

Table 1. Bond lengths involving Cu(II) metal center in Cu-Nap dimeric form. 58

Table 2. Disc diffusion method experimental insets for the naproxenate complexes 78
(Pt-Nap and Cu-Nap) and their starting materials (CuCl22H2O, NaNap and cis-
[PtCl2(DMSO)2]), and results.

Table 3. Antiproliferative effect expressed as concentration required to elicit 50% 85

growth inhibition (GI50) in μg/mL for NaNap, CuCl2‧2H2O, Cu-Nap, cis-
PtCl2(DMSO)2 and Pt-Nap.

Table 4. Antiproliferative effect expressed as concentration required to elicit 50% 85

growth inhibition (GI50) in μg/mL for set 2 (K2PtCl4, Hyd, Pt-Hyd).

Table 5. Antiproliferative effect expressed as concentration required to induce total 86

growth inhibition (TGI) in μg/mL for Cu-Nap and CuCl22H2O, Hyd and Pt-Hyd.

Table 6. Selectivity index (SI) for Pt-Nap, Cu-Nap, Hyd and Pt-Hyd relatively to 87
HaCaT cytotoxicity.

Table A1: EPR spectroscopic parameters of Cu-Nap in the solid-state at room

103
temperature and 77K.
ABBREVIATIONS, ACRONYMS AND SYMBOLS

Main compounds of this work Other chemical compounds

Nap Naproxenate DMSO Dimethylsulfoxide
NapNa Sodium naproxenate DMF Dimethylformamide
Cu-Nap Cu(II) complex with naproxenate MeOH Metanol
Pt-Nap Pt(II) complex with naproxenate TMS Tetramethylsilane
Hyd Naproxen hydrazide Chemical characterization
Pt-Hyd Pt(II) complex with naproxen ESI- Electrospray Ionization -
hydrazide QTOF-MS Quadrupole time-of-flight -
Mass Spectrometry
TGA/DTA Thermogravimetric Analysis /
Molecular Modelling
Differential Thermal Analysis
B3LYP Beck 3-Parameter, Lee, Yang and ATR-FTIR Attenuated Total Reflectance –
Parr functional Fourier Transform Infrared
Spectroscopy
DFT Density Functional Theory ν Wavenumber (cm-1)
LANL2DZ Los Alamos Nation Laboratory 2 NMR Nuclear Magnetic Resonance
– double zeta
SSNMR Solid-State Nuclear Magnetic
Introduction
Resonance
WHO World Health Organization CP/MAS Cross Polarization / Magic-
Angle Spinning
HSQC Heteronuclear Single Quantum
Coherence
COX Cyclooxygenase HMBC Heteronuclear Multiple Bond
Coherence
NSAIDs Non-Steroidal Anti-Inflammatory δ Chemical shift (ppm)
Drugs EPR Electron Paramagnetic
Resonance
Biological studies
ATCC American Type Culture Collection
BHI Brain-Heart Infusion
FBS Fetal Bovine Serum
CFU Colony-Forming Units
MIC Minimum Inhibitory Concentration
GI50 Growth Inhibition in 50%
TGI Total Growth Inhibition
 SUMMARY

CHAPTER 1. INTRODUCTION 20
CHAPTER 2. OBJECTIVES 29
2.1 Specific Objectives 30
CHAPTER 3. METHODOLOGY 31
3.1 Materials and Methods 32
3.2 Data collected for sodium naproxenate (NaNap) 33
3.3 Synthesis of naproxen hydrazide (Hyd) 33
3.4 Synthesis of Cu(II) complex with naproxenate (Cu-Nap) 36
3.5 Synthesis of Pt(II) complex with naproxenate (Pt-Nap) 36
3.6 Synthesis of Pt(II) complex with naproxen hydrazide (Pt-Hyd) 37
3.7 Antibacterial activity assays in vitro 38
3.8 Cells viability and antiproliferative activity assay over tumor cells 39
3.9 Molecular Modeling 40
CHAPTER 4. RESULTS AND DISCUSSION 41
4.1 Naproxen Hydrazide (Hyd) 42
4.2 Cu(II) and Pt(II) complexes with naproxenate (Cu-Nap and Pt-Nap) 48
4.3 Pt(II) complex with naproxen hydrazide (Pt-Hyd) 66
4.4 Antibacterial activity assays 78
4.5 Cell viability assays and antiproliferative activities 79
CHAPTER 5. CONCLUSION 88
REFERENCES 91
APPENDIX 100
A.1 2D NMR of hydrogen/nitrogen of Hyd. 101
A.2 EPR results for Cu-Nap 103
A.3 Z-Matrices for DFT simulations 104
 20

CHAPTER 1

INTRODUCTION
 21

The search and development of new broad-spectrum antimicrobial agents that

are safe and active against multidrug-resistant bacterial strains are considered priorities for
the World Health Organization (WHO). The main reason that led to microbial resistance
and chronic infections was the misuse of antibiotics not only in humans, but also in livestock
and agriculture used to either prevent or cure diseases[1]. This situation has not only caused
death of millions of people around the world, but also affected the financial system of many
countries[1,2].

On the other hand, the search for new pharmacologically active agents on tumor
cells, which minimize adverse reactions and have high efficacy, has also been the object of
several research groups in Medicinal Chemistry around the world. In 2018, there were an
estimated 9.6 million deaths from cancer, being the cause of one in every 6 deaths. The most
common types of cancer in Brazil so far were prostate, breast and colorectal cancers, with
an estimated 130,000 women with breast cancer in 2040, which is alarming[1,3]. More than
20 million new cases of cancer are expected per year by 2025, which proves that discoveries
of new drug treatments and personalized therapies are urgent[3]. Nowadays, it is estimated
that platinum drugs are considered in at least 50% of cancer treatments, but resistance has
also become an issue in this context. This resistance could be related to efflux bomb
mechanism of the tumor cell, or to the presence of thiol proteins in the cytoplasm,
upregulation of anti-apoptotic proteins, or even to a mechanism involving DNA reparation,
among others[4,5]. Additionally, sometimes it becomes necessary to administrate high doses
of the drug to ensure that the right concentration of metal ions would be able to reach the
intracellular medium. This can lead to adverse reactions, such as increased nephrotoxicity,
neurotoxicity, ototoxicity and gastrointestinal reactions, which is the main setback in cancer
treatment[5,6,7].

To overcome these two major challenges in current medicine, the preparation

of bioactive coordination compounds with metals has been considered a promising strategy.
Despite facing resistance in conventional therapies, the application of metals and their
compounds in the history of medicine is well known. In the 1960s, a new era of development
of drugs containing metals began with the discovery of the inhibitory capacity of the
reproduction of Escherichia coli strains by cisplatin and, later, of its inhibitory action on
the proliferation of tumor cells[3,8,9]. Following the great triumph in coordination chemistry,
its derivatives carboplatin and oxaliplatin became widely used in clinic as
chemotherapeutics for ovarian, testicular and melanoma cancer, among others[5]. However,
 22

resistance combined with poor selectivity has also become an issue in therapy with platinum
drugs. Another example is silver sulfadiazine, which has been used since 1970’s in the
treatment of bacterial infections[10]. Besides that, Cu(II) salts and their complexes have also
been investigated for a long time due to its antimicrobial activities[11]. Casiopeínas® are
copper-based compounds (mixed-chelate complexes) that presented inhibitive activity over
Mycobacterium tuberculosis[12] and Trypanosoma cruzi[13]. It was shown that the
Casiopeínas trigger DNA cleavage by free radical mechanism[13]. More recently, complexes
based on Zn(II)[2], Pd(II)[3], Co(II)[14] among other metals are also being explored as
antiproliferative agents.

Metal complexes have unique advantages, such as their redox properties and
reaction mechanisms that differ from organic compounds and can be modulated based on
the different classes of ligands[15]. It is well known that transition metal complexes can act
on different cellular targets simultaneously, hindering the emergence of bacterial and
tumoral resistance to their uses[16]. In Figure 1, it is possible to see some of the possible
interactions in eukaryotic cellular media. After passing through the cell membrane, the
metal complex can interact with thiol-containing proteins and glutathione (GSH), or with
organelles or with the DNA by a timely aquation. They can also difficult the DNA
reparation system. In addition, some metal complex can be photoactivated, being used for
photodynamic therapy[17]. Over bacteria (Figure 2), some of the mechanisms are similar
such as interaction with DNA (helicoidal and plasmid), enzymes and organelles (ribosome,
in this case)[18]. A few metal complexes and metallic materials can cause generation of
superoxide and oxidative radicals (ROS) that can act in the cell membrane and in the inner
cell media. In this context, they can interrupt electron-transport and cause cell wall
leakage[18,19]. Furthermore, metal complexes can be modelled based on the central ion
charge and ligand characteristics to become more lipophilic or hydrophilic, depending on
their applications[15,20]. The choice of ligands that can minimize ligand exchanges with the
external media, such as σ-donors, -acceptors or chelates, is also a valuable strategy[8,11]. In
addition, metal ion complexation can facilitate the transport of the ligand the transport of
organic ligands through the lipidic membrane into the cellular media[21].

A wide variety of ligands can be used, and some drugs already used on clinic
can be associated to metals. As an example, many complexes based on non-steroidal
anti-inflammatory drugs (NSAIDs) were already reported in literature. Several Cu(II)
 23

complexes have been studied in combination with the NSAIDs mefenamic acid, diclofenac,
diflunisal, flufenamic acid and naproxen, showing better anti-inflammatory activities in
vitro than their parental molecules[15]. A synthesized Cu(II)-nimesulide complex, for
example, presented a good minimum inhibitory concentration (MIC) of 3.0 mmol L -1
against Staphylococcus aureus and 1.5 mmol L-1 against E. coli and Pseudomonas
aeruginosa[22].

Figure 1. Mechanisms of action of transition metal complexes over tumor eucaryotic

cells. Adapted from Marlove et al [17].
 24

Figure 2. Mechanism of action of metal complexes over bacteria. Adapted from

Mikhailova[18] and Bogdan et al[19].

Both cancer and bacterial infections are related to defense mechanisms and
promote an inflammatory response in the body, thus associating anti-inflammatories to
metals with known biological activity seems to be an efficient proposal. The combination
of antibiotics with anti-inflammatories is already a widespread practice (polytherapy).
Meanwhile, in cancer cases, the progression and metastasis of tumor cells is related to the
high levels of prostaglandins (PG). These are lipids that have hormone-like effects and are
related to high levels of cyclooxygenases (COXs), which are for instance the main
responsible for the inflammation process. Particularly, COX-2 modulates the expression of
programmed death-ligand 1 (PDL1), that mediates the immune response in
tumorigenesis[23]. In addition, tumor occurrence and proliferation relate to a high expression
of matrix metalloproteinases (MMPs) in acidic and reductive microenvironment,
responsible for degradation of the basement membrane and increase of growth factors. In
this context, inhibitors of COX-1 and COX-2, and possibly of MMPs as well, such as non-
steroidal anti-inflammatory drugs (NSAIDs) has been studied for the treatment of
cancer[5,24].
 25

As an example, a naproxen derivative synthesized by Deb et al. inhibited COX-

2 and consequently stopped the production of PGs, reducing inflammation and hindering
the migration of breast cancer tumor cells in vitro[25]. Recently, our research group used this
approach in the synthesis of Pt(II) and Pd(II) complexes with the hydrazide of ibuprofen,
which is another NSAID[3]. The Pd(II) complex had a selective action on ovarian tumor
cells[26]. Furthermore, mono, binuclear and trinuclear Cu(II) complexes in combination with
NSAIDs such as mefenamic acid, diclofenac, diflunisal, indomethacin, aspirin and
naproxen, among others, are reported in the literature[15,27,28]. Some of these complexes
showed better biological activities in vitro than their parental molecules. For example, a
trinuclear copper(II) complex with naproxen, salicylhydrazide and 1,10-phenanthroline
showed partial intercalation with the DNA, which is not possible when working with free
organic ligands[27]. Nunes et al[22] synthesized Cu(II)-nimesulide complex, for example,
presented minimum inhibitory concentration (MIC) of 3.0 mmol L-1 against Staphylococcus
aureus and 1.5 mmol L-1 against E. coli and Pseudomonas aeruginosa, while nimesulide
alone did not have antibacterial potential[22]. Several platinum (IV) prodrugs with NSAIDs
were reported by Spector et al[29] and Tolan et al[30], Chen et al[5] and many of them
presented higher antiproliferative activity than pure cisplatin.

Naproxen (nap), or (+)-(S)-2-(6-methoxinaphtalen-2-yl)propanoic acid, is a

NSAID with composition C14H14O3. Its structure is shown in Figure 3a. It has been largely
used in clinic to treat arthritis, spondylitis, gout and other rheumatic diseases, bursitis, acute
and chronic pain[14,21]. As an analgesic and antipyretic drug, it has shown less unwanted
cardiovascular and nephron side effects when compared to other NSAIDs[30]. It is a non-
selective NSAID and has an independent mechanism of action by blocking
cyclooxygenases (COX) 1 and 2, involved in the production of prostaglandins. It also has
shown inhibition of MMPs, especially when associated to Pt(IV)[5]. Naproxen has been
reported to reduce carcinogenesis and proliferation of prostate, colon and bladder cancer
tumor cells by inhibiting protein kinase B phosphorylation and inducing cellular
apoptosis[5,30,31].

There are reports in the literature about naproxenate based complexes, proving
that these compounds can present useful biological activities. In literature zinc, cobalt,
copper, and iron complexes with naproxen were reported[32,33,34]. There are studies related
to the synthesis of these metal complexes and the evaluation of its anti-inflammatory,
antioxidant[35], antimicrobial[9] and inhibiting cell proliferation[5,33] activities. They have
 26

presented positive effects on blood glucose and on the central nervous system[34]. A Cu(II)
complex with naproxen previously published in the literature showed moderate antibacterial
activity against several strains of Bacillus sp. (Gram-positive), Shigella sp. (Gram-negative)
and E. coli (Gram-negative)[9]. In special, three binuclear copper(II) naproxenate complexes
call attention with a paddle-wheel type coordination and biological properties. The
structures of [Cu2L4(H2O)2] where proposed for the ligands (L) as the NSAIDs diclofenac,
ibuprofen and naproxen based on infrared (IR) and Raman spectroscopic analysis and the
crystal structure for diclofenac complex[36]. In another study, the [Cu2Nap4(H2O)2]
presented good binding affinity to bovine (BSA) and specially to human serum albumin
(HSA) proteins, which take part in the transport of the metal complexes through the blood
stream[37]. A [Cu2Nap4(DMSO)2] metallodrug which presented antitumor properties was
loaded to chitosan beads was characterized and this system showed a controlled release in
gastric and intestinal pH[38].

Still, most reports of naproxenate based complexes are compounds with diverse
3d transition metals (e.g. Co[14], Cu [39]
, Zn[40]) associated to nitrogen donor heterocyclic
ligands (1,10-phenanthroline, pyridine, 2,2′-bipyridine, etc). They have been reported as
antitumoral agents[41], or antibacterial[21], keeping the anti-inflammatory and antioxidant
properties of naproxen. As an example, a zinc(II) complex with naproxen and 1,3-
diaminopropane exhibited good anti-inflammatory and analgesic potential, and also
antibacterial activity over Gram-positive bacteria S. aureus ( MIC = 56.25 μg/mL) and
Bacillus subtilis (MIC = 112.5 μg/mL)[40]. The monomeric Cu(II) complex
[CuNap2(H2O)3](H2O) presented higher anti-inflammatory response and lower gastric
ulceration potential than free naproxen, working as urease inhibitors[31]. In this context,
other metals such as Pd(II)[7], Pt(II)[7,42] and Ru(II)[41] has also been associated to naproxen
and a N donor ligand. Pt(II) naproxenate complexes with ethylenediamine and
diaminocyclohexane presented selective antiproliferative activity against epithelial (MCF7)
and human cervical carcinoma cells (HeLa), respectively[42,43]. A Pt(IV)-based naproxen
complex was reported as an antitumor agent in vitro and in vivo over CT-26 homograft
tumors in BALB/c mice, being comparable with the known drug oxaliplatin[5].
Ruthenium(II) combined with naproxen and bipyridine or phenanthroline was capable of
binding to DNA and showed in vitro antiproliferative activity over MCF7 and prostate
(PC3) cancer cell lines[41].
 27

Figure 3. Schematic representation of (a) naproxen; (b) naproxen hydrazide.

Furthermore, modifications on the carboxylic moiety found in most NSAIDs

can impact various chemical aspects such as reactivity and lipophilicity, as well as increase
their affinities for specific metals[26]. Many of these ligands have biological activity, such
as isonicotinohydrazide and derivatives that have antidepressant, antitumor and tuberculosis
activities. From a chemical point of view, a hydrazide (nitrogen donor) is a slightly less
hard base according to Pearson's concept than the carboxylic group (oxygen donor), which
can increase the affinity of the ligand to soft acids such as Pt(II) ions and stabilizing the
complex[44]. The preparation and application of hydrazides and especially hydrazone
complexes based on NSAIDs is well established in the literature and has already been
applied in our group for nalidixic acid[45] and ibuprofen[3].

The application and combination of naproxen hydrazides (Figure 3b) with

organic ligands to generate novel hydrazones and Schiff bases has already been addressed
in literature[46,47]. A carbonyl hydrazide can be attached to other molecules that present (or
not) biological activity, by acid condensation with ketones and aldehydes, producing
carbonyl hydrazones[45]. The formation of hydrazones[48] is presented for a general
naproxen-based hydrazone in Figure 4. Hydrazones have been studied as novel
antimicrobial, anticonvulsant, antitumoral, anti-inflammatory and antiviral agents[45].
Naproxen based hydrazones are reported in literature for various biological applications.
One of the arylidenehydrazone derivatives of naproxen synthesized by Azizan et al
presented higher anti-inflammatory activity than naproxen itself. Most of the compounds in
this work binds more to COX-2 than to COX-1, thus being less aggressive for
gastrointestinal tract, in theory[47]. A naproxen-based N-acylhydrazone also was reported to
 28

act as a COX-2 inhibitor[49]. Eight derivatives of naproxen hydrazones with 1,2,4-triazole

ring possessed anticancer activity against prostate (PC-3, DU-143 and LNCaP) tumor cell
lines[26]. Furthermore, naproxen and ibuprofen-based hydrazones showed spontaneous
binding to trypsin in a thermodynamic study[46]. Naproxen based Schiff bases, aroyl and
sulfohydrazides and oxadiazoles showed in vitro urease enzyme inhibitory activity[50].
Finally, copper and cobalt derivatives of naproxen-based Schiff bases showed antibacterial
activities and binding to COX-1[35].

Nevertheless, a detailed physical-chemical characterization or description of the

antibacterial activity of naproxen hydrazide is yet to be reported, as well as its metal
complex with Pt(II).

Figure 4. Schematic representation of the hydrazone formation.

In this context, this work intends to unify the chemical and biological
characteristics of naproxen and its hydrazide with the well-known antiproliferative activities
of metals such as Pt(II) and Cu(II), aiming to study their behavior as antibacterial and/or
antitumor agents.
 29

CHAPTER 2

OBJECTIVES
 30

This work aims to synthesize and characterize Pt(II) and Cu(II) complexes with
naproxen and naproxen hydrazide. The antibacterial and antitumoral activities are also
envisaged.

2.1 Specific objectives

To achieve the main objectives, the following specific objectives were

completed.

• Spectroscopic and physical-chemical characterization of commercial sodium

naproxenate for further comparison;

• Synthesis and characterization of Cu(II) and Pt(II) complexes with naproxenate;

• Synthesis and characterization of naproxen hydrazide;

• Synthesis and characterization of Pt(II) complex with naproxen hydrazide;

• Evaluation of the antibacterial activities of naproxen hydrazide and synthesized

complexes;

• Evaluation of the antitumoral potential of naproxen hydrazide and synthesized

complexes over a panel of tumoral cells;

• Evaluation of the cell viability and selectivity index of naproxen hydrazide and
synthesized complexes over non-tumoral cells.
 31

CHAPTER 3

EXPERIMENTAL
 32

3.1 Materials and Methods

Sodium naproxenate (NaNap), CuCl2·2H2O and Cu(NO3)23H2O, and the

complex cis-[PtCl2(DMSO)2], where DMSO is dimethylsulfoxide, were purchased from
Sigma-Aldrich Laboratories. Elemental analyses were performed on a Perkin Elmer 2400
CHNS/O Analyzer. Infrared spectroscopic measurements (FTIR) were performed on
Agilent Cary 630 and 660 FTIR spectrometers, using the attenuated total reflectance (ATR)
method. The FTIR spectra were recorded from 100 to 4000 cm-1, with resolution of 4 cm-1.
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. Thermal stability
of the complexes was analysed by using a TGA/DTA simultaneous analyser SEIKO
EXSTAR 6000. The compounds were evaluated in oxidant atmosphere using -alumina
crucible, from 25 to 900°C and heating rate of 10 °C/min. The 1H, 13C and the {13C,1H}
HSQC and HMBC {13C,1H} nuclear magnetic resonance (NMR) spectra of the ligands and
Pt(II) complexes were recorded in a Bruker Avance III 500 MHz (11.7 T) spectrometer.
The kinetic study of the Pt(II) complexes was conducted in a Bruker Avance III 400 MHz
(9.4 T) NMR spectrometer. Samples were analysed at 298 K. The naproxenate was analysed
in D2O, while naproxen hydrazide and the Pt(II) complexes were dissolved in DMSO-d6,
due to their better solubility in this solvent. The chemical shifts were given relative to
tetramethylsilane (TMS). Solid state 15N NMR (SSNMR) were recorded in a Bruker Avance
II 400 MHz (9.395 T) spectrometer using the combination of cross polarization, proton
decoupling and magic angle spinning techniques (CP/MAS) in a frequency of 10 kHz.
Electrospray ionization mass spectrometry (ESI-MS) were conducted using a Q-Orbitrap.
The solutions were directly infused into the instrument's ESI source and analysed in the
positive mode, with capillary potential of 3.50 kV. The dilution used was of 50x with
MeOH:H2O 1:1 v/v, adding, when necessary, a second injection with 0.1% of formic acid.
When direct preparation in methanol was not possible due to low solubility of the material,
a solubilization in DMSO was done previously to the dilution in MeOH:H2O. The Cu(II)
complex with naproxenate was further characterized by Electron Paramagnetic Resonance
(EPR) spectroscopy in a CW-Bruker spectrometer, model EMX, X-band (9.5 GHz, 20.12
mW, 100kHz modulation frequency) in solid state, and in frozen DMSO solutions. Samples
were analysed at low temperature (77 K), in Wilmad quartz tubes (4 mm internal diameter),
using a quartz Dewar with liquid nitrogen. DPPH (’-diphenyl--picrylhydrazyl) was
 33

used as frequency calibrant, with g = 2.0036. Simulations of spectra were performed with
EasySpin software at MATLAB environment.

3.2 Data collected for sodium naproxenate (NaNap)

1
H NMR (500 MHz, D2O) δ (in ppm) 1.39 (d, J=7.17, 3H) assigned as H3, 3.67
(q, J=7.42, 1H) H2, 3.80 (s, 3H) H14, 4.70 (s) H2O/D2O, 7.05 (dd, J=8.80, 1H) H10, 7.15
(s, 1H) H8, 7.37 (dd, J=8.65, 1H) H5, 7.62 (s, 1H) H13, 7.66 (d, J=8.38, 1H) H6 that
overlaps with 7.69 (d, J=8.92, 1H) H11; 13
C NMR (500 MHz, D2O) δ (in ppm): 18.27
assigned as C3, 48.43 C2, 55.30 C14, 106.10 C8, 118.21 C10, 125.24 C13, 126.80 C5,
126.96 C6, 128.88 C12, 129.33 C11, 132.93 C7, 138.92 C4, 156.53 C9, 183.91 C1. These
attributions follow the scheme in Figure 5.

14

9 8
7 6
10

11 5
12
4
13
2 3

Figure 5. Numbered structure of NaNap.

3.3 Synthesis of naproxen hydrazide (Hyd)

Based on the synthesis of ibuprofen hydrazide, performed in our research

group[3], naproxen hydrazide was prepared. Initially, 252 mg (1.0 mmol) of sodium
naproxenate was dissolved in a mixture of 12 mL of chloroform, 150 μL of methyl
chloroformate and 200 μL of triethylamine in an ice bath for 20 minutes under constant
stirring. The former increases solubilization in chloroform as it reacts with naproxenate.
When the solution became clear, 250 μL (5.0 mmol) of hydrazine hydrate were added. It
was left to stir in an ice bath for at least 5 minutes and removed from the ice bath. A reflux
system is attached since many volatile compounds were included. After stirring at room
 34

temperature for 3 hours, the mixture was heated in a silicone bath at 100°C for 2.5 hours.
Then, the system was kept under stirring at room temperature for another 22 hours. The
reaction was quenched with 10 mL of deionized water, causing a phase separation. To
improve this separation, sodium chloride was added to this mixture. A liquid-liquid
extraction is performed. The aqueous phase was discarded, and sodium sulphate was added
to remove small amounts of water from the organic phase. After filtration, the organic phase
was left evaporating at room temperature until completely dry. After 24 hours, the formation
of a crystalline solid was observed. The solid was kept in a desiccator under P2O5 and stored
for further characterization. The yield was calculated considering one molecule of
naproxenate for one of hydrazine hydrate, which was placed in excess to optimize the
reaction yield. The reproducibility of the material was verified under the same experimental
conditions in seven replicates. This synthesis is schematized in Figure 6. Recrystallization
of the product is optional, but it was proceeded twice. This purification process consisted in
heating a methanolic solution of the compound, followed by slow cooling, filtration under
vacuum and drying under P2O5. Anal Calcd. for C14H16O2N2 (%): C, 68.80; H, 6.60; N,
11.47. Found (%): C, 68.24; H, 6.49; N,10.88. Yield 83.70%. MW: 244.29 g/mol. 1H NMR
(500 MHz, DMSO-d6) δ (in ppm) 1.40 (d, J=7.13, 3H) assigned as H3, 2.50 solvent residual
peak (DMSO), 3.30 (water content from DMSO), 3.65 (q, J=6.82, 1H) H2, 3.86 (s, 3H)
H14, 4.29 (s, large, 2H) H16, 7.11 (dd, J=9.14, 1H) H10, 7.31 (s, 1H) H8, 7.45 (dd, J=8.49,
1H) H5, 7.77 (s, 1H) H13, 7.79 (d, J=8.92, 1H) H6, 7.83 (d, J=8.95, 1H) H11, 9.22 (s, 1H)
H15; 13
C NMR (500 MHz, DMSO-d6) δ (in ppm): 18.80 assigned as C3, 40.02 DMSO,
43.69 C2, 55.59 C14, 106.15 C8, 119.05 C10, 125.70 C13, 125.70 C5, 126.96 C6, 128.80
C12, 129.54 C11, 133.61 C7, 137.67 C4, 157.44 C9, 173.34 C1; 15N {15N,1H}HSQC NMR
(500 MHz, DMSO-d6) δ (in ppm): 130.90 N15, 55.10 N16; 15
N SSNMR (400 MHz,
CP/MAS 10kHz) δ (in ppm): 137.00 N15, 64.30/54.00 N16. These attributions follow the
enumeration schematized in Figure 7.
 35

Figure 6. Scheme of the experimental procedure of Hyd synthesis.

 36

Figure 7. Numbered scheme of the molecular form of Hyd.

3.4 Synthesis of Cu(II) complex with naproxenate (Cu-Nap)

Firstly, 42 mg (0.25 mmol) of CuCl22H2O were weighed and dissolved in 5.0

mL of deionized water. This solution was added dropwise to 5.0 mL of an aqueous solution
containing 126 mg (0.50 mmol) of sodium naproxenate and the reaction was maintained
under constant stirring for 2 hours at room temperature (23ºC). The solid compound formed
was then filtered under vacuum and the precipitate was washed with distilled water. After
complete drying of the material in a desiccator with P2O5, the precipitate was weighed, and
the reaction yield was calculated considering two molecules of naproxenate for one Cu(II).
The reproducibility of the material was verified under the same experimental conditions in
triplicate. Anal Calcd. for CuC28H28O7 (%): C, 62.27; H, 5.23. Found (%): C,62.24; H, 5.42.
Yield 79.04%. Additionally, the same procedure was performed by using Cu(NO3)2‧3H2O
as starting material and the elemental analysis was similar to the first method. Anal Calcd.
for CuC28H28O7 (%): C, 62.27; H, 5.23. Found (%): C,61.93; H, 5.40. Yield 71.32%.

3.5 Synthesis of Pt(II) complex with naproxenate (Pt-Nap)

Initially, 105 mg (0.25 mmol) of cis-[PtCl2(DMSO)2] was dissolved in 5 mL of

DMSO. This solution was added dropwise to 10 mL of an aqueous solution containing 126
mg (0.50 mmol) of sodium naproxenate and kept under constant stirring for 45 hours at
room temperature. The solid compound formed was then filtered under vacuum and washed
with distilled water. After complete drying of the material in a desiccator with P 2O5, the
 37

precipitate was weighed and the reaction yield was calculated, considering two molecules
of naproxenate for one Pt(II). The reproducibility of the material was verified under the
same experimental conditions in triplicate. Anal Calcd. for PtC32H38O8S2 (%): C,46.33; H,
4.86. Found (%): C,47.46; H, 4.73. Yield 72.61%. MW: 809.85 g mol-1. 1H NMR(500 MHz,
DMSO-d6) δ (in ppm): H11 7.74 (d, J = 8.9 Hz, 1H), H6 7.68 (d, J = 8.5 Hz, 1H), H13 7.57
(s, 1H), H5 7.32 (dd, J = 8.6 Hz, 1H), H8 7.26 (s, 1H), H10 7.13 (dd, J = 6.3 Hz, 1H), H14
3.85 (s, 3H), H2 3.54 (q, J = 7.2 Hz, 1H), H2O/DMSO-d6 3.33, coordinated DMSO/DMSO-
d6 2.55 (s, 6H), DMSO/DMSO-d6 (solvent) 2.50, H3 1.28 (d, J = 7.2 Hz, 3H); 13C NMR(500
MHz, DMSO-d6) δ (in ppm): C1 179.03, C9 157.40, C4 137.94, C7 133.51, C11 129.54,
C12 128.87, C6 127.15, C5 126.88, C13 125.80, C10 118.92, C8 106.28, C14 55.58, C2
47.05, DMSO/DMSO-d6 39.74, C3 19.41. These attributions follow the enumeration
schematized in Figure 5.

3.6 Synthesis of Pt(II) complex with naproxen hydrazide (Pt-Hyd)

Firstly, 50 mg (0.125 mmol) of K2PtCl4, were dissolved in 9 mL of deionized

water. Then, this solution was added dropwise to a solution containing 60 mg (0.25 mmol)
of naproxen hydrazide in 4.5 mL of methanol. The reaction was kept under stirring for 16
hours at room temperature. Then, the white solid obtained was filtered under vacuum and
washed with methanol and deionized water. The solid was dried in a desiccator with P2O5
and the yield was calculated considering two molecules of hydrazide for one Pt(II). The
reproducibility of the material was verified under the same experimental conditions in
triplicate. These attributions follow the enumeration schematized in Figure 5. Anal Calcd.
for PtC28H34O5N4Cl2 (%): C, 43.53; H, 4.44; N, 7.25. Found (%): C, 44.07; H, 4.41; N, 7.22.
Yield 72.50%. MW: 772.58 g mol-1. 1H NMR (500 MHz, DMSO-d6) δ (in ppm) 1.43 (dd,
J=6.9 Hz, 3H) assigned as H3, 2.51 (t) DMSO/DMSO-d6, 3.35 (s, large) H2O/DMSO-d6,
3.71 (q, J=7.2 Hz, 1H) H2, 3.87 (s, 3H) H14, 5.77 (s, large, 2H) H16, 7.13 (dd, J=9.1 Hz,
1H) H10, 7.28 (s, 1H) H8, 7.43/7.47 (dd, J=8.4 Hz, 1H) H5, 7.72 (s, large, 1H) H13,
7.73/7.75 (d, J=8.4 Hz, 1H) H6, 7.77/7.79 (dd, J=8.8 Hz, 1H) H11, 10.36 (s, 1H) H15; 13C
NMR (500 MHz, DMSO-d6) δ (in ppm) 18.76/18.61 assigned as C3, 40.10 DMSO, 43.59
C2, 55.62 C14, 106.14 C8, 119.09 C10, 125.80 C13, 126.93 C5, 127.07 C6, 128.79 C12,
129.55 C11, 133.65 C7, 137.364 C4, 157.48 C9, 173.35 C1; 15N {15N,1H}HSQC NMR(500
MHz, DMSO-d6) δ (in ppm) 137.10 N15; 15N SSNMR (400 MHz, CP/MAS 10kHz) δ (in
ppm) 144.00 N15, 9.50 N16.
 38

3.7 Antibacterial activity assays in vitro

The complexes containing naproxenate and naproxen hydrazide were first

evaluated for its antibacterial activity against Gram-positive (S. aureus and B. cereus) and
Gram-negative (E. coli and Pseudomonas aeruginosa) strains. The method chosen was the
disc diffusion one (antibiogram), following the protocols described in the literature[51]. The
bacterial strains were inoculated in tubes containing BHI (Brain Heart Infusion FASVI) and
incubated for 18h at 35-37 °C. The agar was swabbed following the directions a, b, c and d
(Figure 8) in this order. Sterile filter paper discs (Whatman 3 with 10.0 mm in diameter)
were impregnated with 50 μL of the metal complexes and ligands in triplicate (Table 1).
The amount of compound in each disc ranged from 0.96 to 1.55 mg. The impregnated discs
were then placed on the surface of the solid agar inoculated with a suspension on the
McFarland nephelometric scale 0.5 (~ 1.5 × 108 CFU/mL) of S. aureus (ATCC 25923), B.
cereus (ATCC 14579), E. coli (ATCC 25922), and P. aeruginosa (ATCC 27853) using a
sterile cotton swab. The plates were incubated for 18 h at 35 °C. The sensitivity of the
complexes was evaluated from the diameter of the zones of inhibition (in millimeters). In
Figure 8 it is schematized the method of preparation of the disc diffusion assays, where C-
is the negative control (solvent), C+ is the positive control (CuCl22H2O) and 1 and 2 are
the compounds tested.

Figure 8. Scheme of Disc Diffusion method.

 39

3.8 Cells viability and antiproliferative activity assay over tumor cells

The antiproliferative assays were divided into two experimental sets. In the first
the naproxenate complexes and their starting materials: Pt-Nap, Cu-Nap, NaNap, cis-
[PtCl2(DMSO)2], CuCl22H2O, were investigated on five human tumor cell lines U251
(glioma), NCI-ADR/RES (ovarian expressing phenotype of multiple drugs resistance), 786-
O (kidney), PC-3 (prostate) and HT-29 (colon adeno-carcinoma) and on the non-tumor cell
line HaCat (human keratinocyte) used for cell viability test. The second set included the
complex Pt-Hyd and its starting materials, Hyd and K2PtCl4, which were evaluated on all
lines mentioned before and also on the tumor cell line MCF-7 (breast) and non-tumor cell
line 3T3 (mice fibroblasts). The tumor cell lines were kindly provided by Frederick Cancer
Research & Development Center, National Cancer Institute, Frederick, MA, USA. The non-
tumor cell line HaCat (human keratinocyte) used for cell viability was provided by Dr.
Ricardo Della Coletta (UNICAMP).

Stock cultures were grown in 5 mL of RPMI-1640 supplemented with 5% fetal

bovine serum (RPMI/FBS 5% Gibco1, USA) at 37°C in 5% CO2 with a 1% penicillin :
streptomycin mixture (Nutricell1, Brazil, 1000 U/mL) (complete medium)[52,53]. The
compounds evaluated were dissolved in DMSO (1.0 mg material : 10 μL DMSO).
Afterwards, 50 μL of this solution was diluted in the medium (5000 μg/mL) to 1.0 mL and
then diluted sequentially in 10x, affording the final concentrations of 0.15, 1.5, 15 and 150
μg/mL. Thus, the concentrations tested in μmol/L for the synthesized compounds were: for
Hyd, 614.0, 61.4, 6.14 and 0.61; for Cu-Nap, 138.9, 13.8, 1.38 and 0.14; for Pt-Nap, 185.2,
18.5, 1.85 and 0.18; for Pt-Hyd, 194.1, 19.4, 1.94 and 0.19. For the starting materials in
μmol/L: cis-[PtCl2(DMSO)2], 355.2, 35.5, 3.55 and 0.36; CuCl22H2O, 879.9, 88.0, 8.80
and 0.88; K2PtCl4, 361.4, 36.1, 3.61 and 0.36; NaNap, 594.7, 59.5, 5.95 and 0.59. DMSO
(0.10%) was used as negative control and doxorubicin and cisplatin as positive control for
the same concentrations in μg/mL.

Cells in 96-well plates (100 µL cells/well, inoculation density: 3.5 to 6x104

cell/mL) were exposed to different concentrations of sample and controls (100 mL/well) in
triplicate, for 48 h at 37°C and 5% of CO2. Before (T0 plate) and after the sample addition
(T1 plates), cells were fixed with 50% trichloroacetic acid (50 μL/well), and cell
proliferation was determined by the spectrophotometric quantification of cellular protein
content using the sulforhodamine B assay[54]. The measurements were taken at 540 nm in a
 40

VersaMax Molecular Devices spectrometer. The GI50 values (concentration that inhibits
50% cell growth or cytostatic effect) and TGI values (concentration that inhibits completely
the cell growth) were determined through non-linear regression, type sigmoidal, analysed
using Origin 9.0 software (OriginLab Corporation, USA)[55]. The selectivity index was
calculated based on the GI50 values of non-tumoral HaCaT cells and GI50 of the tumor cell
lines, according to the equation below:

In Figure 9 this assay is schematized, where C- is the negative control

(medium), Cc- is a negative control of the compounds, C+ is a positive control
(doxorubicin) and 1 to 5 are the compounds, tested in triplicate for the 4 concentrations
specified above.

Figure 9. Scheme of a panel of tumoral cells assay.

3.9 Molecular Modelling

Gaussian 09[56] and GaussView[57] programs were used for the computational
simulation and to generate the structures of the compounds, respectively. Density functional
theory (DFT) was used to evaluate the complexation of platinum and copper metal atoms
with sodium naproxenate ligand, Cu-nap and Pt-nap complexes. For the simulations, the
functional M06-2X[58] with 6-31+G(d,p)[59-61] basis set for hydrogen, carbon, oxygen and
sulfur atoms and LANL2DZ[62] effective core basis set for copper and platinum atoms were
used. Frequency calculations were performed, and no imaginary frequency was found,
which confirms that the optimized structures were at minimum energy.
 41

CHAPTER 4

RESULTS AND DISCUSSION

 42

4.1 Naproxen Hydrazide (Hyd)

The hydrazide was synthesized from naproxenate and hydrazine hydrate

following the reaction schematized below (unbalanced partial chemical equation). Hyd
appeared as a non-hygroscopic, yellowish crystalline powder precipitate, coming from the
organic phase of the mixture formed during the quenching on the experimental procedure.
The synthesis was reproducible and showed an average yield of 83.7%, which fits the rate
obtained for hydrazides and organic compounds in the literature in general[3].
Recrystallization crystals matched the NMR data for the precipitate and smaller
light-yellow crystals were obtained. The compound was soluble in methanol, acetone,
acetonitrile, dichloromethane (DCM), chloroform and dimethylsulfoxide (DMSO) at a
concentration of 5 mg/mL.

𝑁𝑎𝑝(𝑠𝑜𝑙) + 𝑁2 𝐻4 (𝑠𝑜𝑙) 𝑁𝑎𝑝𝑁2 𝐻3 (𝑠𝑜𝑙)

Hyd composition suggested by elemental analyses matched the expected

composition for naproxen hydrazide, C14H16O2N2. The Q-Orbitrap ESI-MS spectrum
presented two main peaks at m/z 245.1284 and 169.1101. The former corresponds to the
protonated form of the hydrazide. This indicates once again that the solid obtained is in fact
naproxen hydrazide, which does not undergo considerable fragmentation and, therefore, is
relatively stable under the conditions imposed during the measurements. The presence of
dimers (or polymers) was insignificant near the monomer’s abundance and its main
fragments. The mass spectrum of Hyd is presented in Figure 10.

To better evaluate the coordination sites, the exact group to which a metal could
coordinate to, infrared absorption spectroscopic measurements in solid state (ATR-FTIR)
of Hyd were performed. The spectrum (blue) is presented in comparison with naproxenate
spectrum (orange) in Figure 11a. The typical N-H stretching appeared around 3210 cm-1,
which corresponds to the hydrazine portion. It can also be noticed the appearance of other
bands characteristic of the C=O and C-O stretching modes from 1653 to 1212 cm-1, as well
as C-N bands in Hyd spectrum below 900 cm-1, pointed out in Figure 11b. Altogether, the
synthesis of naproxen hydrazide was confirmed by this technique.

Hyd was further characterized by uni- and bidimensional NMR spectroscopies.

Each signal was attributed to one atom of the suggested molecule, based on shielding
 43

effects, electronegativity, hydrogen coupling and chemical equivalence. The 1H and 13

C
spectra of NaNap in D2O and Hyd in DMSO-d6 are presented in figures 12 and 13,
respectively. The bidimensional HSQC and HMBC{15N,1H} NMR spectra are presented in
15
figures A1 and A2, respectively, while the N SSNMR is presented in Figure 14. NMR
attributions are presented in subsection Chapter 3.

Even though different solvents were used for Nap and Hyd NMR measurements
not allowing a direct comparative between their spectra, a valuable difference was seen for
two signals in 13C NMR spectrum. For Hyd, C1 signal was seen at 173.31 ppm, while for
Nap it appears in 183.91 ppm. This gives evidence that the hydrazine hydrate indeed reacted
at this site of the Nap molecule, promoting the substitution of the -COONa group for -
(CO)NHNH2. Another indication of this substitution is the difference observed for C2, as
this carbon is linked to C1 where the modification is occurring. For Hyd C2 signal appeared
at 43.70 ppm, while for Nap it appeared at 48.43 ppm. For the other carbons, there were no
significant changes when compared to naproxenate, as expected. The 1H NMR spectrum
showed a new singlet at 9.22 ppm corresponding to the -NH hydrogen. A small, large signal
centred in 4.29 ppm visibly overlaps with another signal around 4.35, which may refer to
the two equivalent protons H16 of -NH2 in the hydrazine portion of the molecule. This
matches with the expected results based on the literature for ibuprofen hydrazide, with -NH
at 9.13 ppm and -NH2 as a thin signal at 4.17 ppm[3].

In the HSQC {15N, 1H} (500 MHz; DMSO-d6) NMR, the correlation between
N15 at 131.5 ppm and the H15 in 9.22 ppm was observed. Meanwhile, in the HMBC {15N,
1
H} (500 MHz; DMSO-d6) NMR this H15 coupled with the neighbour N16 at 55.12 ppm.
Both signals can also be seen in 15N SSNMR, with similar δ seen for 15N NMR in solution:
N15 at 137.0 ppm and N16 at 64.3/54.0 ppm. These two signals obtained for N16 in
SSNMR could indicate a polymorphism of solid naproxen hydrazide. The same behavior
was observed for ibuprofen hydrazide[3].
 44

Figure 10. Q-Orbitrap ESI-MS (positive mode, 1%DMSO, 200μL/min in MeOH:H2O,

dilution 20% MeOH:H2O, no column, 3.5kV, R=3.5x104) spectrum of Hyd: (a) Full spectrum; (b)
zoom on experimental spectrum; (c) corresponding simulated spectrum.
 45

Figure 11. ATR-FTIR of Hyd (blue) in comparison with Nap (orange) ranging from: (a) 4000 to 500 cm-1; (b) 1800 to 500 cm-1 (zoom).
 46

Figure 12. 1H and 13C NMR (500 MHz, D2O) of NaNap.

 47

Figure 13. 1H and 13C NMR (500 MHz, DMSO-d6) of Hyd.

 48

Figure 14. 15N SSNMR (10 kHz) of Hyd.

4.2 Cu(II) and Pt(II) complexes with naproxenate (Cu-Nap and Pt-Nap)

The Cu-Nap complex was obtained as a light green powder with good solubility
in DMSO, and poor solubility in methanol and chloroform. The obtained solid using copper
nitrate instead of chloride presented the same behaviour on TGA, ATR-FTIR and similar
composition by elemental analyses. This indicates that the anion in the copper salt does not
affect the composition of the complex in the synthetic procedure. The minimal formula
suggested by elemental analyses was CuC28H28O7H2O. Since it has low solubility in most
solvents, dimeric or polymeric arrangements for the complex should also be taken into
consideration. Meanwhile, the Pt-Nap complex presented itself as a white non hygroscopic
powder with solubility in DMSO, dichloromethane, acetone, acetonitrile and chloroform,
and it was poor soluble in methanol. The composition suggested by elemental analyses was
PtC32H38O8S2.

Mass spectrometric analysis of Cu-Nap was conducted to predict the most

probable composition of the complex. The main signals acquired in the positive mode
(Figure 15a) were at m/z 179.0171 ([(SO(CH3)2)2Na]+ seen as [DMSO2+Na]+), 448.0435
([CuC18H25O5S2]+ or [CuNapDMSO2]+), 176.0089, 146.0070, 218.9569, 503.0902, in
descending order of abundance. The main peak successfully identified was for m/z
522.0665 as [CuC28H28O7+H]+, seen as compositions [CuNap2+H+] (z=1) or
 49

[Cu2Nap4+2H+] (z=2). Though there are other m/z fragments such as for m/z=971.1442, it
could not be seen the expected dimeric form [Cu2Nap4(OH2)2+H]+, with or without its water
molecules. In negative mode (Figure 16) some of the main signals were obtained at m/z
169.0658, 134.8654, 185.0910, 229.0869 (Nap-), 459.1814 and 750.1894 ([CuNap3]-). The
intense signal at m/z 169.0658 also appears for Hyd, referring to the degradation of
naproxenate, and the signals at m/z 134.8654 and 185.0910 also appears in naproxen mass
spectrum reported by Zayed et al[63]. Finally, the presence of high m/z signals with
significant abundance in the negative mode and the presence of signals above m/z 522.50
in the positive mode, even though with low abundance, could be evidence of polymeric
forms of Cu-Nap. However, these are unstable and rapidly degrade under ESI experimental
conditions, especially with addition of formic acid.

For Pt-Nap, in the mass spectrometric analysis two main signals were observed
in the positive mode at m/z 231.1011 and m/z 253.0830. They correspond to protonated
naproxen (HC14H14O3)+ and protonated sodium naproxenate [NaC14H13O3+H]+
respectively. The third most intense signal at m/z 185.0958 could be associated to the
rupture of the COOH group from the Nap molecule, according to Zayed et al[63]. There is
also a low intensity signal at m/z 653.1401 that corresponds to [PtNap2+H]+, corroborating
the 1:2 Pt:Nap composition suggested by elemental analyses. The mass spectrum of Pt-Nap
is shown in Figure 15b.

In thermal analysis shown in Figure 17a, it was noticed for Cu-Nap a large
oxidative exothermic event in the range 210 - 453°C that was responsible for two large mass
losses altogether corresponding to 79.32%. This is related to the oxidation of two
naproxenate molecules (Calcd. 78.95%). The residue of 17.25% is attributed to the
formation of copper oxide, CuO (Calcd. 14.74%). It was observed a small loss of 3.43%
over 100°C which indicates loss of one water molecule (Calcd. 3.34%). Because it was lost
over 100°C it was suggested as one coordination water rather than hydration water. For Pt-
Nap (Figure 17b), thermogravimetric analysis indicated the mass loss of 70.61% from 160
to 350°C, which likely corresponds to the oxidation of 2 naproxenate and 2 DMSO
molecules (Calcd. 75.89%). The residue was of 26.11%, which matches the composition of
PtO (Calcd. 26.08%).
50
 51

Figure 16. Q-Orbitrap ESI-MS

(negative mode, 15 μL/min in
MeOH:H2O, 50x dilution 20%
MeOH:H2O, 1%DMSO, no
column, no HCOOH, 3.5kV,
R=3.5x104) spectra of Cu-Nap:
(a) Full spectrum; (b) zoom on
Cu-Nap experimental spectrum;
(c) simulated spectrum for Cu-
Nap.
 52

Figure 17. TG/DTA from 20° to 800°C with increase rate of 10°C per minute of: (a) Cu-Nap;
(b) Pt-Nap.

The main bands on mid ATR-FTIR spectra of Cu-Nap, Pt-Nap and NaNap are
presented in Figure 18a. The far-IR spectra from 100-500 cm-1 of Cu-Nap and Pt-Nap are
presented in Figure 18b. As a complement, the Raman spectra of Cu-Nap, Pt-Nap, NaNap
and cis-[PtCl2(DMSO)2] are presented in Figure 19.

In fact, in the ATR-FTIR spectrum of Cu-Nap (Figure 18a), there is a sharp

signal at 3601 cm-1, which is characteristic of water directly coordinated to the metal
centre[37], being in accordance with the TGA and elemental analysis. Also, there were no
Cu-Cl bonds in the far-infrared spectrum of the complex, proving once more the absence of
 53

chloride in the composition of Cu-Nap[64] (Figure 18c). When comparing the IR spectra of
Cu-Nap and sodium naproxenate (Figure 18a), interesting differences were observed. The
main absorption bands of NaNap and attributions occurred at 3564-3143 cm-1 (νOH, broad
band), 3057-2830 cm-1 (νCH), 1585 cm-1 (νCOOasym) and 1390 cm-1 (νCOOsym).
Meanwhile, the Cu-Nap FTIR spectrum presented a sharp strong band at 3601 cm-1 (νOH)
instead of the broad band observed for NaNap. Other main signals (in cm-1) for Cu-Nap
were: 3064-2800 (νCH), 1554 (νCOOasym) and 1403 cm-1 (νCOOsym). Thus, it was observed
a difference between the νCOO asymmetric and symmetric stretchings ∆(νCOOasym -
νCOOsym) for Cu-Nap of 151 cm-1, whereas for the free ligand it was of 195 cm-1. The
ν(COO-) being a bit lower in the complex than in the free naproxenate, suggested a bridge
bidentate coordination mode of carboxylate to copper[42]. This was already proposed by
Dimiza et al, where a ∆ν value was of 170 cm-1 for the same complex[37]. Furthermore, the
signals below 635 cm-1 are all supressed in the complex indicating a slight change in -OCO
behaviour. This proposition differs this binuclear (or dimeric) paddle-wheel complex from
the former mononuclear copper(II) naproxenate complex [Cu(H2O)3(nap)2]H2O reported
by Chu et al, where Cu(II) is coordinated by 2 molecules of nap in the monodentate mode
and three water molecules plus a hydration water[25,26]. Thus, IR findings indicated that Cu-
Nap structure occurred as proposed previously by Dimiza et al[37] and also by Trinchero et
al[36].

The absence of a broad band around 3400-3600 cm-1 in Pt-Nap spectrum (Figure
18a) reinforces the absence of hydration water in its composition. The most notable change
when comparing the complex and the ligand IR spectra are the shifts observed for
carboxylate stretchings. In Figure 18b it is possible to observe that the asymmetric
stretching varies from ν(COO) 1585 cm-1 in the ligand spectrum to 1637 cm-1 in the
complex. Additionally, the symmetric stretching varies from 1390 cm-1 in the ligand to 1341
cm-1 in the complex. A doublet band is seen at 1144/1129 (s) (νSO) (Figure 18b) which
matches the one reported for cis-[PtCl2(DMSO)2] in literature, attesting presence of DMSO
in the solid structure[65]. These shifts indicate that the naproxenate is coordinated to Pt(II)
in a monodentate form by one of its oxygen atoms[42,66]. The square-planar coordination
sphere of Pt(II) was completed by two DMSO molecules. Evidence of Pt(II) bonding to the
carboxylate is seen in Raman (Figure 19) and far-FTIR (Figure 18c), with the presence of
ν(PtO) stretching. In addition, even though a trans coordination mode could be suggested
 54

Figure 18. ATR-FTIR from (a) 4000-1200 cm-1 of Cu-Nap, Pt-Nap and
NaNap; (b) 1300 to 500 cm-1 of Pt-Nap and NaNap; (c) 500 to 100 cm-1 of
Cu-Nap and Pt-Nap.
 55

to minimize steric hindrance, the far-IR spectrum showed a double band at 453/430 cm-1,
characteristic of a cis isomer according to literature[65].

Figure 19. Raman spectra of the complexes Cu-Nap and Pt-Nap, and their starting
materials NaNap and cis-[PtCl2DMSO2].
 56

When comparing the Raman spectra (Figure 19), it is noticed the presence of
intense bands below 100 cm-1 that appear in Cu-Nap but do not appear in NaNap. The weak
signals from 100-600 cm-1 occurs for Cu-O stretching[67]. The other signals that appear in
Cu-Nap are derived from the naproxenate spectra, with C-O stretching at 522 cm-1 and C-
H stretching at 750 cm-1. For Pt-Nap it is identified in the Raman spectra the Pt-S (Pt-
DMSO) vibration at 480 cm-1 and the shoulder at 452 cm-1, which also appeared for cis-
[PtCl2(DMSO)2]. Thus, the coordination of Pt(II) to the DMSO molecules occurs by the
sulphur atom. It can also be seen in Raman as well as in far-IR spectra of Pt-Nap, but not
on cis-[PtCl2(DMSO)2] as expected, the Pt-O stretch at 523 cm-1 [68]. However, it is not seen
in Pt-Nap the Pt-Cl stretch identified for the precursor in Raman at 349 cm-1 proving the
substitution of both its chlorides.

The UV-Vis spectrum of Cu-Nap exhibits three absorption bands at 240 nm

(seen only in chloroform), 260 and 320 nm (chloroform and dimethylsulfoxide, doublet
bands), which may be attributed to intraligand transitions. A band in the visible region with
maximum at 715 nm is also observed and attributed to d-d transitions and corresponds to
absorption in red, which matches the greenish color observed in the material. This band is
in accordance with the obtained results by Dimiza et al for a paddle-wheel copper(II)
complex with naproxenate previously described, which was obtained in this work by a
different synthetic method[37]. Thus, the kinetic study in UV for ligand exchange was
measured at 260, 275, 320 and 335 nm and is presented in Figure 20. While Cu-Nap is
reported to retain structure in either solid or in solution[37], it could be observed that in
DMSO there was only a minor change in the absorbance behavior throughout 24h,
indicating that some solvent exchange may occur. This can be noticed especially for the
absorbance peaks 260 and 275 nm during the first eight hours.

EPR spectrum in the solid state at room temperature, presented on Appendix

A.2, Figure A3(a) showed a central profile typical of dinuclear or dimeric complexes,
corroborating once more with the literature[37]. Additionally, a radical signal appeared at
350 mT. The spectrum in the solid state at 77K, presented in Figure A3(b) showed good
correlation with the simulation as well. An isotropic signal with no hyperfine structure was
obtained. The spectroscopic parameters are shown in Table A1.
 57

Figure 20. Kinetic UV-Vis spectra of Cu-Nap from 0 to 24h in DMSO.

Finally, in order to evaluate the proposed structure for Cu-Nap, molecular

modeling studies were conducted. The minimum molar composition of 1Cu(II) : 2Nap :
1H2O suggested by elemental analysis, TGA and mass spectrometric data was considered
in the construction of this structure. It was observed that a paddle-wheel binuclear form,
inspired in the literature[69] is the most probable to exist in the solid state. A polymer with
compositions [Cu2Nap4]n was already reported by Abuhijleh et al, where the units are linked
via Cu-carboxylate interactions from neighboring units[70]. As discussed above Cu-Nap was
proposed in a dimeric/polymeric structure, with a bidentate bridging coordination mode of
carboxylate to Cu(II). According to literature, the water molecules should be placed in
apical sites of binuclear Cu(II) paddle-wheel structures[71]. Thus, polymerization would
occur by the water molecules, with a Cu-Ow interaction, where Ow refers to oxygen of
water molecules. In this context, the paddle-wheel structure schematized in Figure 21a was
proposed for Cu-Nap, as it was already suggested for Cu(II) complexes combined to
NSAIDs published by Trinchero et al[36] and Mikuriya et al[69]. DFT simulations for the
binuclear (dimeric) form are presented in Figure 21b and the bond lengths are presented on
Table 1. The results show that the distance between the copper atoms (Cu1-Cu2) is 2.870
Å and that the bonding distance between the oxygen atoms of the ligands and the Cu atoms
 58

(Cu-O) ranges from 1.872 Å to 2.556 Å. This is in accordance with the values reported in
the literature for Cu(II) paddle-wheel complexes[31,67].

Table 1: Bond lengths involving Cu(II) metal center in Cu-Nap dimeric form.

Bond Distance (Å) Bond Distance (Å)

Cu1-O1 2.295 Cu2-O2 1.875

Cu1-O3 2.124 Cu2-O4 1.888

Cu1-O6 2.134 Cu2-O5 1.895

Cu1-O8 2.556 Cu2-O7 1.872

Cu1-Ow1 2.158 Cu2-Ow2 2.343

Cu1-Cu2 2.870

On the other hand, further investigation was made for Pt-Nap, using 1D and 2D
NMR techniques, acquiring spectra for both the complex and the free ligand. The 1H and
13
C NMR for the complex (Figure 22) was evaluated, in accordance with the attributions
made in NaNap spectra (Figure 12). The 195Pt NMR (500 MHz, DMSO- d6) is presented in
Figure 22. The HSQC and HMBC {13C,1H} NMR (500 MHz, DMSO-d6) spectra of Pt-Nap
are shown in figures 23 and 24, respectively. The shifts are numbered according to the
attributions for the naproxenate and DMSO that consists in the organic portion of Pt-Nap,
according to the schematized molecule on the top.

One of the most remarkable particulars from the 1H NMR spectra of the
complex is the presence of two signals for DMSO. The signal at 2.50 ppm (quintet) is
attributed to the residual peak of the solvent (CHD2(SO)CD3) used in the experiment. The
other signal at 2.55 ppm is referred to the DMSO bonded to platinum (singlet,
CH3(SO)CH3). So, the coordination of DMSO to Pt(II), which was suggested before by
infrared spectroscopic measurements, is confirmed by NMR. Furthermore, no significant
shifts (∆δ) were seen when comparing 1H spectra of the complex and the ligand, which was
expected since the modification was proposed to be on C1 that is not bonded to any proton
itself.
 59

Figure 21. (a) Structural formula for the Cu-Nap complex; (b) Molecular model for Cu-Nap.

On the other hand, the 13C NMR spectrum of the Pt-Nap complex exhibited a
significant shift of the carbon atom of the carboxylate group in Pt-Nap when analysing both
 60

ligand (183.91 ppm, D2O) and complex (179.03 ppm, DMSO-d6) spectra. This reinforces
the coordination of Pt(II) by this group, corroborating the Raman observations. Only a small
shift was seen for C2, which was expected since this carbon atom is bonded to the
carboxylate group. Thus, the former cis-[PtCl2(DMSO)2] used in the synthesis would have
the substitution of both its chlorides for naproxenate molecules. This was evidenced in 195Pt
NMR, with the shift of -347.4 ppm relatively to the signal for cis-[PtCl2(DMSO)2]. In other
words, there is less shielding effect and narrower electronic density around Pt(II) in Pt-Nap
than in its starting complex. During the assignment on the molecule a better definition of
the aromatic C5 and C13 was seen in the HMBC {13C, 1H} ligand spectrum (Figure 24),
while in the HSQC {13C, 1H} NMR spectrum of the complex it was easier to distinguish H6
from H11 as shown in Figure 23.

Moreover, 195Pt NMR spectroscopic data were also acquired for Pt-Nap (Figure
25) and its starting Pt(II) complex, cis-[PtCl2(DMSO)2]. Potassium hexachloridoplatinate
(IV), K2PtCl6 was used as reference, being in accordance with the procedure reported by
Quintanilha et al in literature[72]. For the precursor, it was obtained a single peak at δ = -
3452.8 ppm, which was similar to the value of δ= - 3445.96 found by Quintanilha et al. For
Pt-Nap, it was observed a single peak at δ= -3105.4 ppm, with delta ∆δ= - 347.4 ppm (low
field) in comparison to the single peak observed for its precursor. In other words, there is
less shielding effect and narrower electronic density around Pt(II) in Pt-Nap than in its
starting complex. This could be in part explained by the slight increase in electronegativity
when substituting chloride (z=3.16) in cis-[PtCl2(DMSO)2] by oxygen (z=3.44) of the
COO- group of Nap, which is the coordination site in Pt-Nap. As oxygen is more
electronegative it attracts electronic density to itself, thus leaving Pt(II) less shielded. It is
worth mentioning that the chelating effect cannot be taken into consideration for Pt-Nap,
since it was concluded that Nap would be coordinated to Pt(II) in the monodentate form.
Regardless, the presence of a single 195Pt peak for Pt-Nap proves that the sample consisted
in only one species of a Pt(II) complex, and altogether with the 1H and 13C NMR results it
can be assumed that there was no presence of impurities in the final product of the synthesis.
 61

Figure 22. (a) 1H NMR (500 MHz, DMSO-d6) of Pt-Nap; (b) 13C NMR (500 MHz, DMSO-d6) of Pt-Nap.
 62

Figure 23. HSQC {13C, 1H} (500 MHz, DMSO-d6) of Pt-Nap.

 63

Figure 24. HMBC {13C, 1H} (500 MHz, DMSO-d6) of Pt-Nap.

 64

To evaluate ligand exchange between Pt-Nap and DMSO solvent a kinetic study
using NMR was made with measurements from t = 0 to 24h. As seen in Figure 26, there
was no shift or change in the 1H NMR spectrum during the time, which confirms the stability
of this complex in DMSO. This is a positive point when considering biological assays in
prokaryotic or eukaryotic cells.

Figure 25. 195Pt NMR (500 MHz, DMSO-d6) spectra for Pt-Nap (top) and cis-
[PtCl2(DMSO)2] (bottom).

Based on all results, it was possible to suggest that Pt-Nap would be in fact
[Pt(Nap)2(DMSO)2], with coordination on a monodentate mode of two Nap molecules and
2 DMSO and absence of Cl or hydration water. Indeed, this coordination mode was also
reinforced by the DFT studies. The most stable structure of Pt-Nap was observed to be
square-planar with Pt(II) coordinated directly to sulphur atom of 2 DMSO molecules.
Analyzing the bonds length for the trans [Pt(Nap)2(DMSO)2] and cis [Pt(Nap)2(DMSO)2]
configurations, it is possible to observe that the Pt-O and Pt-S bonds length are very close
between the structural configurations and the values are in accordance with the distances
typically found in complexes in the literature[66].
 65

Figure 26. Kinetic 1H NMR(500 MHz, DMSO-d6) spectrum of Pt-Nap in t = 0h, 0.5h,
1h, 2h, 4h, 6h, 12h and 24h.

The electronic energy values were calculated at the M06-2X/6-

31+G(d,p)/LANL2DZ level for the two geometrical conformations. It was possible to
determine that the energy difference between the complexes cis and trans is 0.9 kcal mol-1,
indicating that both possibilities may occur. Both models are presented in Figure 27. It
should be noted that Pereira et. al[73] when studying the formation of amantadine,
rimantadine and memantine complexes with platinum(II) [PtCl2(atd)(DMSO)],
[PtCl2(mtn)(DMSO)] and [PtCl2(rtd)(DMSO)] obtained results of X-ray diffraction that
showed the cis configuration for the complex [PtCl2(mtn)(DMSO)] and trans for
[PtCl2(atd)(DMSO)] and [PtCl2(rtd)(DMSO)]. Together with the X-ray data, the theoretical
results found, MP2/6-31+G(d,p)/LANL2DZ and B3LYP/6-31+G(d,p)/LANL2DZ, showed
that both cis and trans isomers were equally possible to occur because the energy difference
between the complexes was less than ± 2.0 kcal·mol-1, and the same applies to the present
work. However, 195Pt NMR showed a single peak, and 1H and 13C NMR spectra were clean,
suggesting that only one geometric isomer would be possible. With an insight on Raman,
far and mid IR spectra while comparing Pt-Nap to cis-[PtCl2(DMSO)2], also found in
 66

literature[65,68], the peaks corresponding to the cis form could be identified for Pt-Nap, this
being the only isomer thus obtained.

Figure 27. Molecular models of Pt-Nap isomers: (a) trans [Pt(Nap)2(DMSO)2]; (b) cis-
[Pt(Nap)2(DMSO)2].

4.3 Pt(II) complex with naproxen hydrazide (Pt-Hyd)

The Pt-Hyd complex presented itself as a beige non hygroscopic powder with
solubility in DMSO and dimethylformamide (DMF) at room temperature. The composition
suggested by elemental analyses was PtC28H34O5N4Cl2. In mass spectrometric analysis
(Figure 28) the main signals were observed at m/z 594.1055, m/z 760.2125, m/z 179.0171,
m/z 307.1415 and m/z 245.1283. The latter corresponds to (HydH)+, [C14H16N2O2+H]+.
However, the identification of the other fragments corresponding to Hyd is yet to be defined.
The isotopic pattern of platinum expected for the protonated Pt-Hyd in the composition
proposed before for z=1 would be around m/z 772.59 if the hydration water was taken in
 67

Figure 28. Q-Orbitrap ESI-MS ((positive mode, 1%DMSO, 200 μL/min in MeOH:H2O, dilution 20% MeOH:H2O, no column, 3.5kV, R=3.5x104) of Pt-Hyd: (a)
Full spectrum; (b) zoom of Pt-Hyd experimental spectrum; (c) simulated spectrum for Pt-Hyd.
 68

account and m/z 754.57 if it was not. However, the molecular ion appeared at m/z 760.2125,
which for instance, could be interpreted as the composition of Pt-Hyd with presence of
lithium, [Pt(Hyd)2Cl2+Li]+ at m/z 760.1606. The presence of lithium is quite common in
MS and might be derived from impurity on the solvents used in Pt-Hyd analysis. No
coordination to MeOH or presence of formic acid were identified. No presence of either the
protonated form of naproxen or of its sodium salt was found as well, as expected since Hyd
mass spectrum did not present these two fragments either. In addition, it was suggested that
Pt-Hyd is unstable in its dimeric form since the signal at m/z 1515.3580 (orange arrow)
which could be related to the composition [Pt2Hyd4Cl4+Li]+ (m/z 1516.0810) appear with
very low intensity. This was also expected since the structure proposed for Pt-Hyd as well
as its molecular modelling simulations ruled out the possibility of a polymerization. The
full mass spectrum of Pt-Hyd is shown in Figure 28a, in which HydH+ and
[Pt(Hyd)2Cl2+Li]+ are pointed out. On Figure 28b, it is shown the comparison of
experimental and calculated data for the latter fragment.

Thermogravimetric analysis of Pt-Hyd (Figure 29) showed two main

exothermic events: one of 38.82% of mass loss (DTA peak 184-248 ºC) and another of
32.71% of mass loss (DTA peak 357-505ºC), which together sum up to 71.53%. This value
corresponds to the loss of two Hyd molecules (Calcd. 63.24%) and two Cl (Calcd. 9.19%),
that were coordinated to Pt(II) (Sum Calcd. 72.43%). A small loss of 1.98% below 100ºC
may refer to the presence of one hydration water (Calcd. 2.32%). The residue at 700ºC was
of 26.84%, which is suggested as PtO (Calcd. 27.97%). The final mass loss of 7.31%
observed between 700ºC and 900ºC corresponds to the reduction of PtO to Pt residue (Exp.
19.53%) with loss of oxygen (Calcd. 7.57%). Thus, the adopted composition of
PtHyd2Cl2H2O, which matches elemental analysis data, was confirmed by the TGA.
 69

Figure 29. TG/DTA of Pt-Hyd, from 20° to 900°C with increase rate of 10°C per minute.

Infrared spectrum of the Pt-Hyd complex (Figure 30a) shows an absorption

band around 3500 cm-1 relative to OH stretching, (OH), which could indicate presence of
hydration water in the analysed sample, being in accordance with the TGA data. When
comparing the FTIR spectra of the complex and the ligand, the only meaningful change
noticed was the suppression of some signals such as in 1560, 999, 815 and 657 cm-1 that
appeared in the ligand spectrum, which are pointed out in Figure 30b. These signals are
related mostly with C-O stretches. The absence of remarkable changes was expected since
the ligand was proposed to be intact in its coordination to Pt(II). To complete the square-
planar geometry, typical for platinum(II) complexes, and to guarantee neutrality, two
molecules of Cl from the precursor would be directly coordinated to Pt(II)[3].

When comparing solid and solution NMR spectra of the complex and the ligand,
valuable information was acquired on where Pt(II) would be coordinated, and attribution
was made according to Hyd spectra. The 1H NMR spectrum of the Pt-Hyd complex is shown
in Figure 31 (bottom) in comparison with Hyd spectrum (top), both in DMSO-d6. The 13C
NMR spectra in DMSO-d6 for the complex (bottom) and the ligand (top) is shown in Figure
32. The HMBC {13C,1H}, is shown in Figure 33. The HSQC {15N,1H} in DMSO-d6 is
shown in Figure 34. The 15N SSNMR is shown in Figure 35.
 70

Wavenumber (cm-1) Wavenumber (cm-1)

Figure 30. ATR-FTIR of Hyd (blue) and Pt-Hyd (beige) from (a) 4000-500 cm-1; (b) 1800-500 cm-1 (zoom).
 71

Figure 31. 1H NMR (500 MHz, DMSO-d6) of Pt-Hyd (bottom) comparatively to Hyd (top).
 72

Figure 32. 13C NMR (500 MHz, DMSO-d6) of Pt-Hyd (bottom) in comparison to Hyd (top). The vertical lines in Pt-Hyd spectrum refer to the signals that
indicate mixture of products, likely isomers.
 73

Figure 33. HMBC {13C, 1H} (500 MHz, DMSO-d6) of Pt-Hyd.

 74

Figure 34. 2D NMR (500 MHz, DMSO-d6) HSQC {15N, 1H} of Pt-Hyd.

Figure 35. 15N SSNMR (10 kHz) of Pt-Hyd.

A significant shift of 1.15 ppm to low field was noticed for H15, which would
indicate interaction of Pt(II) solely with the hydrazine part of Hyd. With magnification of
 75

the 1H spectrum in DMSO-d6 it was possible to attribute H16 with a shift of ∆δ = -1.48 in
comparison to Hyd. This reaffirms that Pt(II) coordinates mainly with NH-NH2 group.
When analysing the 13C spectra of the complex and the ligand, no visible shifts were seen,
proving once more that there was no coordination of Pt(II) to the carbonyl group of Hyd.
However, some unexpected 1H and 13
C signals were obtained (orange vertical lines in
Figure 32) suggesting that the synthesis probably resulted in a mixture of compounds.
Unexpected 1H signals with small intensities at 7.56 and 9.60 ppm also appeared in Pt-Hyd,
but not in Hyd. The 13C signals at 136.54, 42.90 and 20.66 ppm where not identified in Hyd
spectrum either. Since the 13C signals are very next to C1, C2 and C3 attributed signals, and
these are the carbons closer to the coordination site, they could be referring to an isomer
form of the identified Pt-Hyd. Shift of the signal next to C1, named C1’, was of only C1’-
C1= 0.89 ppm. In this mindset, C2’-C2= 0.57 ppm and C3’-C3= 1.90 ppm, considering the
orange lines marked on Figure 32. The shifts are very low and thus could be referring to
geometric isomerism cis/trans. Even so, the HSQC and HMBC {13C, 1H} showed no
interaction among these non-attributed 13C and 1H signals nor with the Pt-Hyd 13C and 1H
expected signals, thus the most abundant isomer/compound Pt-Hyd could be correctly
identified. In HMBC it was possible to verify the interaction of C1 solely with H15 at 10.36
ppm, and not with the mixture protons that appear above 9 ppm, as it is pointed out in Figure
33.

The HSQC {15N, 1H} most intense signal (Figure 34) suggested that the nitrogen
N15 at 137.1 ppm coupled with the hydrogen at 10.36 ppm, instead of the one at 9.64 ppm,
thus there was noticed a shift of -1.14 ppm relative to H15 in the ligand Hyd. Yet, a mixture
was indicated again by duplicated H/N signals at 9.37/128.8 and 10.37/134.4 ppm. The
former signal might correspond to residual naproxen hydrazide used in the synthesis of Pt-
Hyd, which presents a signal in 9.22/130.9 ppm or to the -NH of the other isomer form.
15
Nevertheless, it was possible to obtain signals for both N15 and N16 in N SSNMR at
144.0 and 9.50 ppm, respectively (Figure. 35). A shift of ∆δ=+54.8 ppm of N16 in Pt-Hyd
SSNMR relatively to Hyd suggested coordination of Pt(II) to this nitrogen instead of N15,
being in accordance with the literature[3]. The SSNMR signals of Pt-Hyd are not as well
defined as for Hyd, what could be explained by the existence of two isomers in solid state.

The cis and trans configurations were theoretically evaluated by molecular

modeling and the results of the structural parameters are shown in Figure 36. The bond
lengths in Ångström (Å) for the complexation are highlighted in figures 36a and 36b for the
 76

trans and cis form, respectively. The results found for the bond lengths of the complexes
are close in both configurations with values of Pt–Cl = 2.370 Å and Pt–N = 2.081 Å for the
trans [Pt(Hyd)2(Cl)2] complex; and Pt–Cl = 2.359Å and Pt–N = 2.097 Å for the cis
[Pt(Hyd)2(Cl)2] complex. The bond length values reported for the Pt(II) complex of
ibuprofen hydrazide by Manzano et al were near to those found for Pt-Hyd, being 2.308 Å
Pt-Cl and 2.049 Å Pt-N[3]. The values of electronic energies for the two conformations were
also evaluated and the difference between the complexes was 3.60 kcal mol -1. The results
show that by the theory employed DFT/M06-2X/6-31+G(d,p)/LANL2DZ both
configurations can occur, reinforcing the possibility of Pt-Hyd being a mixture of both
forms. The matrixes used in the simulations of Pt-Hyd and the naproxenate complexes are
presented in Appendix A.2.
 77

Figure 36. Molecular models of Pt-Hyd isomers: (a) trans-[Pt(Hyd)2Cl2]; (b) cis-[Pt(Hyd)2Cl2].
 78

4.4 Antibacterial activity assays

No significant antibacterial activities were seen for naproxen, its hydrazide or

to Pt-Nap, Cu-Nap and Pt-Hyd complexes. The strains were also resistant over all starting
materials in the considered experimental conditions. In Figure 37a a representative picture
of each strain is shown. In Figure 37b no inhibition halo is seen for both naproxenate
complexes when using the disc diffusion method, only the starting material CuCl22H2O,
also used as positive control. In Figure 37c no inhibition halos were seen for K2PtCl2, Hyd
and Pt-Hyd. The experiment followed the insets explicit on Table 2.

Table 2. Disc diffusion method experimental insets for the naproxenate complexes (Pt-Nap and Cu-
Nap) and their starting materials (CuCl22H2O, NaNap and cis-[PtCl2(DMSO)2]), and results.

Compounds Inhibition zone (mm)

Concentration Mass in
Molecular Weight
(μg/µL) disc (mg) S. aureus B. cereus E. coli P. aeruginosa
(g/mol)

CuCl22H2O 11.0 14.0 11.0

30.98 1.549 NI
170.48 (+0,52) (+1,03) (+0,30)

NaNap
21.76 1.088 NI NI NI NI
252.24

cis[PtCl2(DMSO)2]
27.06 1.353 NI NI NI NI
422.25

Cu-Nap (dimer)
19.24 0.965 NI NI NI NI
1080.12

Pt-Nap
20.08 1.042 NI NI NI NI
809.85

K2PtCl4
27.52 1.376 NI NI NI NI
415.09

Hyd
10.65 1.065 NI NI NI NI
244.29

Pt-Hyd
32.94 1.647 NI NI NI NI
772.58
 79

Figure 37. Disc diffusion of Pt-Nap, Cu-Nap, NaNap, cis-[PtCl2(DMSO)2] over: A) S. aureus,
B) B. cereus, C) E. coli, D) P. aeruginosa.

4.5 Cell viability assays and antiproliferative activities

The complexes and the starting materials were submitted to antiproliferative

activity assays over bacterial strains and a panel of tumoral cells. It is important to note that
Cu-Nap seems to present only a small ligand exchange in DMSO, especially during the first
8 hours (Figure 20) and Pt-Nap has shown stability in DMSO throughout 24h (Figure 26).
This probably has not affected the stability on cellular media and in fact no precipitation or
undesired color change was observed during experimental procedure. The antiproliferative
 80

activity over tumor and non-tumor cells procedure was performed in two separate sets as
explained in the methodology (Section 3.8). The first set included the naproxenate
complexes (figures 38d and 38e) and their starting materials (figures 38a, 38b, 38c), while
the second set, Hyd and its Pt(II) complex, followed by its Pt(II) precursor (Figure 39).

Cu-Nap (Figure 38d) presented a remarkable cytostatic effect over all tested cell
types, especially against NCI (74%) and HT29 (86%) and even presented a low cytocidal
activity over U251 (27%), at the highest concentration of 150 μg/mL (138.9 μmol/L). In
particular, the GI50 over U251 was of 33.9 μg/mL (31 μmol/L) and TGI of 105.9 μg/mL
(98.0 μmol/L). This behavior is most probably due to the presence of Cu(II) ions since
CuCl2·2H2O was cytocidal against all tumor cells evaluated (Figure 38a), especially against
the lines U251, NCI/ADR-RES and 786-0 in which over 50% of the cell growth was
inhibited.

On the other hand, when applied Pt-Nap at 150 μg/mL (185.2 μmol/L) it
presents the highest inhibition for all cell types, as expected. The highest antiproliferative
activity was obtained for U251 (glioblastoma) at this concentration, nearing 48%. For
NCI/ADR-RES (ovarian multidrug resistant), 786-0 (renal) and PC-3 (prostate) inhibition
was also over 40% and for HT29 (colon) it was over 35%. Interestingly, Pt-Nap had a more
pronounced activity than its starting materials against 786-0 and PC-3. The biological
activities can be considered moderate under the experimental conditions. Even though Pt-
Nap had a more pronounced activity than its starting materials against 786-0 and PC-3, the
cytostatic behavior did not surpass 50% of inhibition and GI50 was not determined by this
experimental procedure. As noticed in Figure 38e, Pt-Nap has a similar activity profile to
NaNap (Figure 38c), and the biological activities can be considered modest under the
experimental conditions.

The antiproliferative activity of naproxen hydrazide (Figure 39 b) was higher

than the obtained for NaNap (Figure 38c) even at the lowest concentration of 0.15 μg/mL
(0.614 μmol/L). This cytostatic effect continues to grow when increasing the concentration
of Hyd over HT29, 786-0 and U251. When applied at 150 μg/mL (64.68 μmol/L), Hyd
showed also to be cytocidal to both the latter tumor cell types to a small extent, killing 5%
of 786-0 and 31% of U251.
 81

Pt-Hyd (Figure 39c), for instance, showed higher antiproliferative activity than
Hyd (Figure 39b), and K2PtCl4 (Figure 39a) as well. When applied in higher concentrations
of 15 (19.41 μmol/L) and 150 μg/mL (194.1 μmol/L), there is an abrupt growth of
antiproliferative activity of the complex over all cell types. In this preliminary assay it was
obtained a maximum of 90% inhibition of PC-3 cell proliferation, which is an interesting
result of cytostatic effect. Indeed, at concentration of 150 μg/mL (194.1 μmol/L), Pt-Hyd
induced 13%, 46% and 62% cell death of NCI, HT29 and MCF-7, respectively. For this
concentration the other results were affected by the poor solubility of Pt-Hyd and the
eventual limitations of the experimental procedure, with deposition of a thin film. Thus an
apparent growth in proliferation is seen in the analysis for higher concentrations.
Regardless, there was 89% and 99% cell death of 786-0 and U251, respectively, when
applied Pt-Hyd at 15 μg/mL (19.41 μmol/L) only, showing a remarkable cytocidal effect of
this complex. For 786-0, PC-3 and NCI there seemed to be dual contribution from its
precursors Hyd and K2PtCl4 cytotoxic profile for the antiproliferative behavior of Pt-Hyd,
though it was much more pronounced in the complex. However, for MCF-7 the activity of
the complex diverged completely from its starting materials, which together summed up to
62% inhibition at the highest concentration (150 μg/mL), when Pt-Hyd already inhibited
67% of the proliferation at 15 μg/mL (19.41 μmol/L).

However, cytotoxicity to non-tumor cells turned out to be an issue for most of

the compounds synthesized in this work. In Figure 40 it is shown that both Hyd and Pt-Hyd
have cytocidal effect against the human line of immortal keratinocytes, HaCaT, while Cu-
Nap present a significant cytostatic effect. The antiproliferative activity over HaCaT
became a challenge for Cu-Nap, being highly cytostatic (75% inhibition) at 150 μg/mL
(138.9 μmol/L). Still, at lower concentrations it has low toxicity, even lower than its starting
materials. Fortunately, the same does not occur for Pt-Nap, which presented just a low
reduction of cell growth, even for the highest concentration tested. Pt-Nap seems to follow
NaNap low toxicity behavior, giving even a slightly greater response.

Naproxen hydrazide HaCaT viability profile diverged completely from NaNap,

already reducing cell proliferation above 50% at 15 μg/mL (61.40 μmol/L), and at 150
μg/mL (614.0 μmol/L) it induced cell death over 30%. It also inhibited the proliferation of
normal mice fibroblast 3T3 cell over 27% at higher concentrations (15 and 150 μg/mL).
Thus, it is possible to conclude that for Hyd the inhibition and toxicity potential against
 82

Figure 38. Antiproliferative profile (%) for data in μg/mL of (a) cis-PtCl2(DMSO)2; (b) NapNa; (c) CuCl22H2O;
(d) Cu-Nap; (e) Pt-Nap.
 83

Figure 39. Antiproliferative profile (%) of (a) K2PtCl4; (b) Hyd; (c) Pt-Hyd.
 84

tumor cells in contrast to its cytotoxicity to normal cells was not as great as desired to enable
its immediate use in the clinic, in contrast to naproxen.

Still, from the toxicity point of view, the worst scenario was obtained for Pt-
Hyd complex, which induced 94% of cell death at 15 μg/mL (19.42 μmol/L). Besides, there
was a very expressive inhibition of 3T3 cell proliferation, near 80% at this concentration.
Since Pt-Hyd was the compound which presented the highest activity against the tumoral
cells, this undesired effect became a difficult challenge to be managed if this work is
continued. To overcome toxicity over normal cells creating local delivery systems such as
nanofibers or other controlled systems, for application in skin cancer for example, may be
a route to explore[74,75].

Figure 40. HaCaT cytotoxicity for the synthesized compounds on this work, in
comparison to NaNap.

The concentrations for inhibition of the cell proliferation in 50%, the GI50, is
shown for all the compounds of the first set on Table 3 and for the second set on Table 4.
Since the curves placed themselves mostly above the 50% line, the calculations extrapolate
the experimental data for concentrations above 150 μg/mL in most of the cases. Similarly,
some of the TGI results presented on Table 5, which represent the concentration of the
compound so that all the cells do not grow, also are superior to 150 μg/mL. Thus, it was
only presented here the results for the compounds which occupied the inferior quadrant on
 85

the plots: Cu-Nap and CuCl22H2O, due to the copper activity and Pt-Hyd and Hyd, due
mainly to the ligand activity, as discussed previously.

Table 3. Antiproliferative effect expressed as concentration required to elicit 50% growth inhibition
(GI50) in μg/mL for NaNap, CuCl2‧2H2O, Cu-Nap, cis-PtCl2(DMSO)2 and Pt-Nap.
NaNap CuCl22H2O Cu-Nap * Pt-Nap
μg/mL μg/mL μg/mL μg/mL μg/mL
(μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L)
>150 27.0 ±0.1 33.9±0.1 >150 >150
U251
(>594.7) (~158.4) (~31.4) (>355.2) (>185.2)
NCI/ADR >150 17.5 ±0.1 44.3 ±62.76 >150 >150
-RES (>594.7) (~102.6) (~41.0) (>355.2) (>185.2)
>150 22.7 ±0.1 >150 >150 >150
786-0
(>594.7) (~133.1) (>138.9) (>355.2) (>185.2)
>150 36.2 ±0.1 147.0 ±0.1 >150 >150
PC-3
(>594.7) (~212.3) (~136.1) (>355.2) (>185.2)
>150 25.2 ±0.1 58.1±0.1 >150 >150
HT29
(>594.7) (~147.8) (~53.8) (>355.2) (>185.2)
> 150 44.7 ±0.1 86.0 ±37.7 >150 >150
HaCaT
(>594.7) (~262.2) (~79.6) (>355.2) (>185.2)
*cis-[PtCl2(DMSO)2]

Table 4*: Antiproliferative effect expressed as concentration required to elicit 50% growth inhibition
(GI50) in μg/mL for set 2 (K2PtCl4, Hyd, Pt-Hyd).

K2PtCl4 Hyd Pt-Hyd

μg/mL μg/mL μg/mL
(μmol/L) (μmol/L) (μmol/L)
>>150 15.8 ±0.1 4.4 ±0.1
U251
(>>361.4) (64.68) (5.69)
>150 >150 6.0 ±9.9
MCF7
(>361.4) (>614.0) (7.77)
NCI/ADR >150 38.5±48.7 2.1 ±1.7
-RES (>361.4) (157.6) (2.72)
>150 2.3 ±2.5 3.4 ±0.1
786-0
(>361.4) (9.41) (4.40)
>>150 >~150 2.5 ±4.2
PC-3
(>>361.4) (>~614.0) (3.24)
>>150 22.2 ±4.8 11.0 ±0.1
HT29
(>>361.4) (90.88) (14.24)
>> 150 13.0 ±0.1 2.1 ±0.1
HaCaT
(>>361.4) (53.21) (2.72)
14.3 ±20.6 >>150* 3.2±8.2
3T3
(34.45) (>>614.0) (4.14)

** This value is for the extrapolation not considering the result presented for the concentration of 1.5 μg/mL,
which seems to be an outlier and a possible experimental error.
 86

Table 5*: Antiproliferative effect expressed as concentration required to induce total growth
inhibition (TGI) in μg/mL for Cu-Nap and CuCl22H2O, Hyd and Pt-Hyd.

CuCl22H2O Cu-Nap Hyd Pt-Hyd

μg/mL μg/mL μg/mL μg/mL
(μmol/L) (μmol/L) (μmol/L) (μmol/L)
47.98 ±0.1 105.9 ±15.1 64.1 ±12.9 3.2± 0.3
U251
(281.4) (98.0) (262.4) (4.14)
>>150 30.3 ±3.6
MCF7 − −
(>>614.0) (39.2)
NCI/ADR 30.4 ±3.6 >150 >>150 54.1 ±31.3
-RES (178.3) (138.9) (>>614.0) (70.0)
47.2 ±9.0 >>150 115.5±0.1 3.0 ±2.1
786-0
(276.9) (>>138.9) (473.1) (3.88)
98.5 ±0.1 >150 >>150 >150***
PC-3
(577.8) (>138.9) (>>614.0) (>194.1
68.8 ±1.0 >150 >150 17.8 ±11.1
HT29
(403.6) (>138.9) (>614.0) (23.0)
>150 >150 59.5±6.3 4.1±0.1
HaCaT
(>879.9) (>138.9) (243.6) (5.31)
>>150 >150***
3T3 − −
(>>614.0) (>194.1)

*** May be affected by precipitation of Pt(0) film in the plate well, which misleads live cells count.

The selectivity index (SI), expressed by the equation below[76], was used to
verify the selectivity of the compounds towards tumoral and non-tumoral cells. For this, the
values of GI50 in μmol/L presented on tables 3 and 4 were considered. The values of SI
obtained for the synthesized compounds in this work are presented in Table 6.

The values of SI below 1.0, which was the case for most of the reported data,
indicate there is not a selectivity between normal and tumoral cells[44]. In other words, for
values of SI below or equal to 1.0, or near it, the compound would either kill both the
tumoral and the non-tumoral cells. The SI values once more reflects the high toxicity of
Hyd and Pt-Hyd mentioned earlier. On the other hand, for Cu-Nap it was obtained some
selectivity against HT-29 (SI=1.48), NCI-ADR/RES (SI=1.94) and specially over U251 (SI
=2.53). Even though the cytostatic behavior over HaCaT may become even higher for doses
above 150 μg/mL (138.9 µmol/L), Cu-Nap could be further explored for such tumor cells.
Pt-Nap activity over HaCaT was minimal, and it could be estimated that good SI values
 87

could be obtained in higher concentrations, but assays exploring new concentration insets
would be needed.

Nevertheless, tolerance to a high dose of any drug is still a challenge in

medicinal chemistry, so it would be important to further investigate the studied compounds,
especially Pt-Nap and Cu-Nap. New tests in higher concentrations in vitro over other non-
tumoral cells as well as HaCaT for both naproxenate complexes could be made and then
proceed to in vivo testing.

Table 6: Selectivity index (SI) for Pt-Nap, Cu-Nap, Hyd and Pt-Hyd relatively to HaCaT
cytotoxicity.

CuCl22H2O Cu-Nap Hyd Pt-Hyd

U251 1.65 2.53 0.82 0.48

MCF7 − − − 0.35

NCI/ADR
2.55 1.94 0.34 1
-RES
786-0 1.97 − 5.65 0.62
PC-3 1.23 0.58 − 0.84
HT29 1.77 1.48 0.58 0.19
 88

CHAPTER 5

CONCLUSION
 89

Copper(II) and platinum(II) complexes with naproxenate were obtained.

Compositions of the complexes were determined by elemental and thermal analyses as
CuC28H28O7 and PtC32H38O8S2, respectively. Coordination of nap to Cu(II) occurs by the
oxygen atoms of the carboxylate group in a bridging bidentate mode. Thermal analysis and
ATR-FTIR suggested one water molecule coordinated to each Cu(II), which would be in the
axial position of the paddle-wheel structure. Mass spectrometric data, EPR and molecular
modeling optimization suggested that Cu-Nap would be dinuclear (or dimeric), or even in a
polymeric form, which would explain its low solubility in most solvents. Conversely, infrared
and NMR spectroscopic data suggested a monodentate coordination of two Nap molecules to
Pt(II). The square-planar coordination sphere was completed by two DMSO molecules.

Naproxen hydrazide was obtained successfully with composition C14H16N2O2

confirmed by elemental and mass spectrometric analyses, and it was used in the synthesis of its
complex with Pt(II). The composition of this complex was also inferred from elemental and
mass spectrometric measurements as being PtCl2C28H32N4O4 H2O. The Pt-Hyd complex did
not vary significantly from Hyd when considering the IR spectra but did present a hydration
water. NMR and SSNMR spectra confirmed that -NH2 on Hyd does not lose its hydrogen atoms
upon coordination to Pt(II). The presence of two chlorines completing the typical square planar
structure of the Pt(II) complex and leading to a neutral charge was confirmed by molecular
modeling.

Preliminary antibacterial activity assays showed that the bacterial strains considered
in the experiment were resistant to the complexes synthesized in this work and naproxen
hydrazide under the experimental conditions. However, new antibacterial tests in vitro, such as
MIC in a 96-wells plate or disc diffusion method for higher concentrations are still necessary
to attest the potential of the compounds. On the other hand, the assays with tumoral cells showed
promising results. Pt-Nap did not present inhibition over 50% of the tumor cell lines growth but
presented an even lower toxicity against HaCaT. Cu-Nap presented a more intense cytostatic
effect and some selectivity against HT-29 and PC-3. More importantly, Cu-Nap showed a low
but significant cytocidal effect over U251 tumor cell line, with a GI50 of 79.6 μmol/L and TGI
of 98 μmol/L, and SI=2.53. Furthermore, Pt-Hyd presented an expressive cytostatic effect over
all cell lines when applied in concentrations above 1.94 μmol/L only. It possessed a low
cytocidal effect for NCI/ADR-RES and mild over HT-29 and MCF-7 at 194.1 μmol/L. In
addition, Pt-Hyd had a high cytocidal potential over U251 and 786-0, promoting 99% and 89%
 90

of tumor cell death, respectively, at 19.41 μmol/L. Meanwhile, its ligand precursor Hyd
presented mild cytocidal effect against both tumor cell lines at 150 μg/mL (614 μmol/L), in
opposition to NaNap, which had no effect against tumor cell lines whatsoever. On the other
hand, Hyd and Pt-Hyd were much more toxic to HaCaT than the naproxenate complexes,
presenting cytocidal effect, with Pt-Hyd promoting 94% death on this cell line when applied at
19.41 μmol/L. Besides, there was also a very expressive inhibition of 3T3 cell proliferation,
nearing 80% for Pt-Hyd at this concentration. Thus, differently from Cu-Nap, selectivity for Pt-
Hyd is very low. Since Pt-Hyd was the most active against all tumor types, this undesired toxic
effect would be a challenge to manage in future works. However, local treatment with
controlled delivery systems could circumvent this issue. As Pt-Hyd and Cu-Nap showed
promising results for antiproliferative activities over tumor cells, further studies as the synthesis
of a copper complex with naproxen hydrazide could also be made with this aim. In addition,
much more could be explored from naproxenate derived complexes in the field of other
biological applications, such as antiviral and fungicide activities.
 91

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monography, Universidade Federal de Minas Gerais, 2019.

76. Muller, P. Y.; Milton, M. N. The determination and interpretation of the therapeutic index in

drug development. Nat. Rev. Drug Discov., 2012, 11, 751–761.

 100

Appendix
 101

A.1 2D NMR of hydrogen/nitrogen of Hyd.

Figure A1. HSQC{15N,1H} NMR

(500 MHz, DMSO-d6) of Hyd.
 102

Figure A2. HMBC {15N,1H} NMR (500 MHz, DMSO-d6) of Hyd.

 103

A.2 EPR results for Cu-Nap

Figure A3. EPR results for Cu-Nap in the solid state at: (a) Room temperature; (b) 77K.

Table A1: EPR spectroscopic parameters of Cu-Nap in the solid-state at room temperature
and 77K.
gx gy gz Ax Ay Az
Solid at RT 1.9072 2.0430 2.5026 187G 17G 200G
Solid at 77K giso 2.1780 Aiso 16G
 104

A.3 Z-Matrices for DFT simulations.

Trans-[Pt(Nap)2(DMSO)2]
C -7.59381500 -0.63344600 -1.32356300
C -6.98102800 -0.51282700 -0.09442500
C -5.92998100 0.41255900 0.10006900
C -5.49761300 1.22211500 -0.98910000
C -6.14701400 1.07264000 -2.23870400
C -7.17151200 0.17186800 -2.41321400
H -5.57140700 -0.06640300 2.18849000
H -7.32348800 -1.13491000 0.72730200
C -5.27041700 0.56024300 1.35341500
C -4.43237500 2.14562000 -0.79759000
H -5.82364700 1.68687500 -3.07507400
H -7.64491300 0.08471800 -3.38371900
C -3.81217200 2.27494100 0.42254200
C -4.24635200 1.45691400 1.50535000
H -4.11113600 2.74221900 -1.64774200
H -3.71970800 1.51906300 2.45303100
O -8.59806800 -1.54895500 -1.41813700
C -9.28131800 -1.66615600 -2.64852600
H -10.04587000 -2.42712900 -2.49574000
H -9.76113500 -0.72101700 -2.93002300
H -8.60714000 -1.98692500 -3.45186900
C -2.61929400 3.19543500 0.63059100
H -2.59761300 3.46694500 1.69164200
C -2.62423800 4.45037600 -0.23432400
H -3.56782500 4.99054800 -0.11899200
H -1.80401500 5.11259100 0.05623400
H -2.47506900 4.19968000 -1.28650500
C -1.37212000 2.34255300 0.36441300
O -1.13355400 1.49047100 1.32480000
O -0.71281000 2.44636900 -0.66150700
S -1.75309600 -1.34861700 1.43551300
O -2.56824000 -0.96540000 2.61961600
C -1.37685600 -3.09758900 1.57162400
 105

H -2.31453400 -3.58012200 1.85610600

H -0.99678700 -3.45654300 0.61522900
H -0.63275500 -3.20589700 2.36160400
C -2.72940500 -1.30001400 -0.06751400
H -2.10479900 -1.71267500 -0.86443800
H -3.64378100 -1.87385900 0.10375200
H -2.96879000 -0.25071200 -0.25903700
Pt 0.12728900 -0.06972900 0.96154000
C 7.34806400 0.99240700 -0.98873100
C 7.21726900 -0.10820300 -0.17039100
C 6.08621200 -0.95244600 -0.27604200
C 5.08286200 -0.66259800 -1.24423500
C 5.24340600 0.48123700 -2.06497400
C 6.34449500 1.29492400 -1.94830000
H 6.65256400 -2.30733700 1.31890300
H 7.99432000 -0.31538600 0.55948700
C 5.89264300 -2.07413500 0.57754500
C 3.92884200 -1.48433900 -1.32584900
H 4.45803400 0.72574200 -2.77371500
H 6.43215800 2.16716500 -2.58495000
C 3.74710100 -2.54930800 -0.47540500
C 4.75732500 -2.84072200 0.48109700
H 3.15361200 -1.21090200 -2.03989600
H 4.61524900 -3.68087900 1.15670400
O 8.46584800 1.75166800 -0.81359000
C 8.64374200 2.88233800 -1.64047600
H 9.59441800 3.32377100 -1.34203200
H 8.69094500 2.60053300 -2.69888600
H 7.84248300 3.61651000 -1.49348900
C 2.46464700 -3.35748100 -0.52502300
H 2.47235000 -4.04841900 0.32920000
C 2.31572600 -4.15597000 -1.81958900
H 3.16423100 -4.83477600 -1.94018000
H 1.38900000 -4.73371000 -1.81516300
H 2.28399200 -3.48393500 -2.68115100
C 1.25871800 -2.43683600 -0.29265800
O 1.40744400 -1.65337500 0.74019300
 106

O 0.25459300 -2.47866000 -0.99589900

S 1.99057700 1.19774800 0.35844700
O 2.40599300 1.16645900 -1.06728300
C 3.34632700 0.61353000 1.38197500
H 4.25067600 1.13407100 1.05436100
H 3.10475300 0.82653400 2.42622300
H 3.43151100 -0.46296800 1.21786000
C 1.86818600 2.90255900 0.90144900
H 1.49183100 2.91977000 1.92676000
H 2.86587700 3.34002900 0.82539000
H 1.16820800 3.38077100 0.21664400
 107

Cis-[Pt(Nap)2(DMSO)2]
C -7.27217947 -0.60882881 4.13856167
C -6.11000596 0.06719189 4.44557302
C -5.48033347 0.89048286 3.48572569
C -6.05339916 1.02421760 2.18907024
C -7.24671234 0.31700572 1.90566583
C -7.84958968 -0.48324819 2.84848466
H -3.83771499 1.51867521 4.75675120
H -5.68793226 -0.04035602 5.44039076
C -4.27719545 1.60173681 3.76601979
C -5.40960434 1.84040151 1.21718286
H -7.69118557 0.41277407 0.91842023
H -8.76203866 -1.00949277 2.59523549
C -4.23981642 2.50302340 1.50003723
C -3.68026412 2.37615899 2.80579251
H -5.85275563 1.90588677 0.22634531
H -2.74765785 2.89042078 3.02160461
O -7.80643269 -1.38001847 5.12464115
C -9.02388656 -2.04849309 4.86718692
H -9.27585942 -2.57511595 5.78719580
H -9.82418095 -1.33953712 4.62436891
H -8.91765395 -2.77460648 4.05253267
C -3.45545242 3.24449201 0.43157797
H -2.83266979 3.99457129 0.92976633
C -4.30733297 3.89753911 -0.64989087
H -5.05606443 4.55726682 -0.20239391
H -3.67573074 4.48542589 -1.32048713
H -4.80654399 3.14362723 -1.26188386
C -2.50508949 2.19221487 -0.16525229
O -1.51926023 1.90107776 0.63504372
O -2.73719707 1.64284368 -1.23429516
S -1.47266295 -1.22107628 0.56548239
O -1.48105130 -2.35761569 -0.40110452
C -3.15615584 -0.71217040 0.88478341
H -3.70490991 -1.61415235 1.16482150
H -3.15834621 0.03312880 1.68428458
H -3.53402470 -0.28246654 -0.04518320
 108

C -0.94452950 -1.81753794 2.16910143

H -0.99433891 -0.98667026 2.87641624
H -1.60779059 -2.63727002 2.45319943
H 0.08377690 -2.16254699 2.03506637
Pt -0.14062380 0.54556229 -0.01546578
C 7.08165795 0.78039023 -4.16671430
C 5.80970586 1.21312552 -4.47781751
C 5.03022153 1.90418529 -3.52364686
C 5.56510203 2.15741852 -2.22850958
C 6.87372024 1.70002562 -1.94080467
C 7.62240162 1.02642854 -2.87809772
H 3.29715285 2.18857918 -4.79797801
H 5.41755657 1.01744257 -5.47144826
C 3.71117579 2.36363928 -3.80826585
C 4.77315620 2.83883863 -1.26223695
H 7.29014979 1.88849341 -0.95467681
H 8.62015089 0.69120636 -2.62167258
C 3.49634935 3.25712785 -1.54908044
C 2.97338037 3.01321069 -2.85338003
H 5.19421871 2.99737212 -0.27228583
H 1.95819373 3.33302178 -3.07226669
O 7.75737859 0.12152209 -5.14743207
C 9.08205479 -0.29344439 -4.88595401
H 9.43296873 -0.76737672 -5.80218864
H 9.72772874 0.56040692 -4.64911080
H 9.11963250 -1.02015195 -4.06578896
C 2.58114176 3.83844636 -0.48556290
H 1.82371788 4.44810679 -0.98884522
C 3.28775078 4.65394054 0.59027156
H 3.89290586 5.44427188 0.13721932
H 2.55271064 5.11161607 1.25696557
H 3.92467733 4.01714850 1.20748375
C 1.85513791 2.62477949 0.11992201
O 0.94601660 2.14003503 -0.67733323
O 2.18964023 2.13971238 1.19293343
S 1.51242058 -0.92996013 -0.58387882
O 1.74306819 -2.03513888 0.39155258
 109

C 3.06353714 -0.10318991 -0.90880428

H 3.77876215 -0.88215812 -1.18227284
H 2.91992647 0.62171147 -1.71414392
H 3.34919785 0.39964655 0.01738352
C 1.11245969 -1.63101693 -2.18226456
H 0.99870728 -0.81210016 -2.89609357
H 1.92379539 -2.30692024 -2.46061063
H 0.17173644 -2.16997472 -2.04451683
 110

Copper complex (Cu-Nap)

Cu -0.17792400 -0.09007200 0.46552400
Cu -0.27566500 0.04498100 -2.39960900
C 5.52123600 4.30694800 1.29234900
C 4.95219000 4.94935700 0.21343000
C 3.58064200 4.78284300 -0.08363100
C 2.77822700 3.95690000 0.75381500
C 3.38959700 3.31753400 1.86035300
C 4.72932700 3.47667900 2.12973000
H 3.57672800 6.00790700 -1.87224500
H 5.58063300 5.57169600 -0.41678700
C 2.97456600 5.37284900 -1.22782500
C 1.41608300 3.74338300 0.42763900
H 2.78206000 2.67893700 2.49756100
H 5.16922100 2.96651800 2.97847800
C 0.85866900 4.28774100 -0.70462600
C 1.66122500 5.11885700 -1.53331700
H 0.82516200 3.08220700 1.05856500
H 1.22098400 5.55196300 -2.42809800
O 6.85254400 4.51316400 1.49011400
C 7.47409800 3.87174700 2.58400400
H 8.52059500 4.17353100 2.55280300
H 7.03348700 4.19023800 3.53642600
H 7.40947300 2.78048000 2.49675100
C -0.57829600 3.97094500 -1.09119400
H -0.75452500 4.34789000 -2.10262200
C -1.59670600 4.56121000 -0.11485700
H -1.48038300 5.64789200 -0.07330900
H -2.62148200 4.32513000 -0.41840700
H -1.44048800 4.15033800 0.88514400
C -0.67692900 2.44292600 -1.13901700
O -0.37395100 1.93436100 -2.28501400
O -0.96290800 1.80662100 -0.11858400
C 5.37384000 -4.52344800 1.86180300
C 5.33535500 -4.42715300 0.48683200
C 5.01135300 -3.20422800 -0.14065800
C 4.73227600 -2.05768700 0.65768600
 111

C 4.78258700 -2.18807300 2.06754000

C 5.08982800 -3.38795500 2.66539200
H 5.14933000 -3.94367400 -2.17368800
H 5.55185000 -5.31043500 -0.10676100
C 4.93154300 -3.07604500 -1.55593700
C 4.39219400 -0.83337900 0.02509900
H 4.56061000 -1.31908200 2.68242300
H 5.11078800 -3.45559800 3.74644200
C 4.28488600 -0.73914800 -1.34229000
C 4.56218200 -1.88886400 -2.13297000
H 4.20083600 0.03771100 0.64972100
H 4.47352300 -1.81432900 -3.21376400
O 5.69056100 -5.74217900 2.37775200
C 5.75667800 -5.87850800 3.78202600
H 6.03826600 -6.91429700 3.96911900
H 6.51453800 -5.21300400 4.21226300
H 4.78502800 -5.67818500 4.24923000
C 3.79820500 0.53358400 -2.00887900
H 3.92775500 0.43190800 -3.08932100
C 4.48319200 1.80724400 -1.51330700
H 5.55944200 1.74575900 -1.69713800
H 4.08474800 2.68417900 -2.03236600
H 4.31582000 1.95962100 -0.44391800
C 2.28838200 0.59182500 -1.72799200
O 1.56828500 0.15197100 -2.70456800
O 1.86209500 0.98128200 -0.64081700
C -5.20542200 -5.18030600 1.54770700
C -4.76776700 -5.59890300 0.30942200
C -3.48909300 -5.22868000 -0.16557000
C -2.64522400 -4.42104800 0.64962400
C -3.12266900 -4.01066300 1.91775600
C -4.37034100 -4.37399100 2.36620500
H -3.65392000 -6.23008700 -2.08121300
H -5.42438500 -6.21291900 -0.29976000
C -3.01797000 -5.61358800 -1.45158400
C -1.37456300 -4.02451100 0.16203800
H -2.48866400 -3.38013800 2.53570600
 112

H -4.70823200 -4.03346400 3.33755800

C -0.94284300 -4.39568900 -1.08917400
C -1.78792300 -5.20301000 -1.89854800
H -0.75417700 -3.38220100 0.78439100
H -1.44834400 -5.49018100 -2.89038500
O -6.45303000 -5.58018900 1.92006400
C -6.93725100 -5.17288200 3.18247200
H -7.93318400 -5.60515800 3.27491200
H -6.30273600 -5.54896000 3.99400100
H -7.00869800 -4.08067200 3.25037100
C 0.40839700 -3.93599100 -1.61066700
H 0.47642200 -4.19559000 -2.67114100
C 1.57716100 -4.54591500 -0.83632700
H 1.54413500 -5.63599300 -0.91819500
H 2.53213200 -4.18320600 -1.22470300
H 1.52026900 -4.27363600 0.22070300
C 0.42431800 -2.40840900 -1.51504300
O -0.17835200 -1.82510500 -2.49462300
O 0.92259700 -1.83402300 -0.54118800
C -5.06918000 5.30125900 1.77477800
C -5.58921500 4.79333600 0.60366000
C -5.27981900 3.47794500 0.18882400
C -4.42665300 2.67082500 0.99471500
C -3.91445100 3.21997800 2.19537800
C -4.22040000 4.50184500 2.58591100
H -6.42590800 3.53774300 -1.65026500
H -6.23490900 5.42352200 -0.00064700
C -5.77337900 2.93080200 -1.02832200
C -4.08795100 1.36323500 0.56366100
H -3.25336200 2.61184900 2.80739500
H -3.80469400 4.89299900 3.50676700
C -4.56760900 0.85582600 -0.62078200
C -5.42268100 1.66356700 -1.41930700
H -3.40585500 0.77306000 1.17293800
H -5.79884000 1.26339800 -2.35756700
O -5.41626300 6.57951000 2.09087400
C -4.88170700 7.14736600 3.26834600
 113

H -5.27822000 8.16108900 3.31823800

H -5.19703100 6.59165700 4.15945900
H -3.78645600 7.18768400 3.23066300
C -4.17858500 -0.53960300 -1.08328000
H -4.52937900 -0.67159400 -2.11095700
C -4.74491800 -1.63755500 -0.18302700
H -5.83512000 -1.55782900 -0.14789300
H -4.47320000 -2.63113000 -0.55220900
H -4.34956500 -1.53648900 0.83023700
C -2.64874300 -0.57247000 -1.11377000
O -2.14841400 -0.07176300 -2.19203500
O -2.00688100 -0.98451200 -0.14098000
O 1.54645900 -0.32199600 1.74141900
H 2.03909000 0.26345500 1.13710600
H 1.79111300 -1.21089800 1.43782600
O -0.86799200 0.43679100 -4.63237600
H -0.70748800 1.38914400 -4.63558200
H -1.81843600 0.33027600 -4.49387300
 114

Trans-[Pt(Hyd)2(Cl)2]
Pt 0.00001300 0.00021900 -1.32054700
Cl 2.32567700 -0.45785800 -1.32258800
Cl -2.32563200 0.45814500 -1.32252500
C -6.46583900 1.60983500 1.39198000
C -5.37840700 1.02273800 2.00417400
C -4.84888100 -0.19123500 1.51787500
C -5.44418300 -0.81519800 0.38615800
C -6.55628500 -0.18772100 -0.22142500
C -7.06423200 0.99601000 0.26225700
H -3.25872900 -0.34115400 2.98449000
H -4.93199700 1.51226400 2.86420400
C -3.72364400 -0.81821900 2.12625900
C -4.89805300 -2.03328300 -0.10563100
H -7.01429100 -0.65317200 -1.09012900
H -7.91625500 1.45035100 -0.22893200
C -3.81503800 -2.62078800 0.49759700
C -3.22599900 -1.99314100 1.63406000
H -5.35078500 -2.49070900 -0.98381700
H -2.35888400 -2.45347200 2.10400900
O -6.90829500 2.77867800 1.92954400
C -8.00451900 3.42530400 1.31767000
H -8.17714600 4.33300800 1.89524200
H -7.77932300 3.69425000 0.27881200
H -8.90528600 2.80069500 1.34760900
C -3.18789400 -3.89320600 -0.04241600
H -3.67731500 -4.13890800 -0.99345900
C -3.30551300 -5.07619700 0.91758300
H -4.35988500 -5.28873700 1.10806200
H -2.82672600 -5.96469900 0.49994100
H -2.81711700 -4.85143400 1.86878100
C -1.72297800 -3.60509700 -0.35436400
O -0.77729300 -4.19139100 0.14175900
N -1.53226000 -2.58416600 -1.26215400
H -2.24023300 -1.85091800 -1.32159500
N -0.21793500 -2.06904800 -1.33553200
H 0.31037500 -2.47316400 -0.55032500
 115

H 0.23247900 -2.42331300 -2.18129900

C 6.46563000 -1.61023600 1.39191500
C 5.37819100 -1.02314300 2.00411300
C 4.84877000 0.19091400 1.51792000
C 5.44416500 0.81496100 0.38630300
C 6.55627200 0.18749800 -0.22128100
C 7.06413000 -0.99631300 0.26230700
H 3.25854700 0.34078400 2.98447300
H 4.93170200 -1.51274700 2.86405700
C 3.72353700 0.81791100 2.12631700
C 4.89813900 2.03313700 -0.10539000
H 7.01435400 0.65301900 -1.08990700
H 7.91617100 -1.45063200 -0.22886800
C 3.81512600 2.62064400 0.49783100
C 3.22599000 1.99290300 1.63420400
H 5.35096100 2.49062700 -0.98349700
H 2.35886400 2.45324000 2.10412800
O 6.90799800 -2.77914700 1.92940100
C 8.00399800 -3.42598000 1.31733600
H 8.17640100 -4.33383500 1.89473700
H 7.77865800 -3.69468700 0.27844800
H 8.90494100 -2.80162800 1.34731600
C 3.18810400 3.89318100 -0.04200900
H 3.67760900 4.13904100 -0.99296900
C 3.30570900 5.07601700 0.91818200
H 4.36007500 5.28851200 1.10874300
H 2.82695700 5.96458300 0.50063400
H 2.81725300 4.85113900 1.86932100
C 1.72318500 3.60522900 -0.35414000
O 0.77749400 4.19150600 0.14198100
N 1.53248900 2.58445400 -1.26211500
H 2.24038800 1.85113300 -1.32158000
N 0.21809900 2.06946500 -1.33570200
H -0.31030900 2.47381500 -0.55068000
H -0.23205600 2.42357500 -2.18166700