Design, synthesis and photobiological activity of novel ruthenium phthalocyanine complexes

Full text of DOI:10.1016/j.inoche.2018.11.004

Accepted Manuscript
Design, synthesis and photobiological activity of novel ruthenium
phthalocyanine complexes
Laisa B. Negri, Tassia J. Martins, Nicholas R. da Silva Gobo,
Kleber T. de Oliveira, Michael R. Hamblin, Roberto S. da Silva
PII:
S1387-7003(18)30384-8
DOI:
Reference:
https://doi.org/10.1016/j.inoche.2018.11.004
INOCHE 7168
To appear in:
Inorganic Chemistry Communications
Received date:
Revised date:
Accepted date:
26 April 2018
24 October 2018
8 November 2018
Please cite this article as: Laisa B. Negri, Tassia J. Martins, Nicholas R. da Silva Gobo,
Kleber T. de Oliveira, Michael R. Hamblin, Roberto S. da Silva , Design, synthesis and
photobiological activity of novel ruthenium phthalocyanine complexes. Inoche (2018),
https://doi.org/10.1016/j.inoche.2018.11.004
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ACCEPTED MANUSCRIPT
Design, synthesis and photobiological activity of novel ruthenium phthalocyanine complexes
Laisa B. Negri1, Tassia J. Martins2, Nicholas R. da Silva Gobo3, Kleber T. de Oliveira3*, Michael
R. Hamblin4,5* and Roberto S. da Silva1,2,4,5*
1Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão
Preto Universidade de São Paulo, Ribeirão Preto, SP-Brazil. *silva@usp.br
2Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto
Universidade de São Paulo, Ribeirão Preto, SP-Brazil
3Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil.
4Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Boston, MA
02114, USA.
5Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA.
Ruthenium phthalocyanine complexes could function as novel photosensitizers for
photodynamic therapy (PDT)1-3. Essentially PDT involves the combination of non-toxic
photosensitizers undergoing irradiation with harmless visible light, that in the presence of oxygen,
lead to the production of singlet oxygen as well other reactive oxygen species (ROS) that can
induce cell death4-6. The cytotoxic efficiency of a photosensitizer is generally governed by the
quantum yield and absorption coefficient, as well as the localization within the cell, because singlet
oxygen can only diffuse a short distance7-9. The ideal photosensitizer should preferentially
accumulate in cancer cells and tumors, which generally depends on the molecular structure of the
photosensitizer. Understanding the relationship between the photosensitizer structure and
cytotoxicity pathways is fundamental for the development of PDT for different types of cancer as
well as non-cancer diseases10. In order to explore the use of ruthenium phthalocyanines as
photosensitizers it is essential to develop synthetic methods to prepare different complexes with
modifications on the phthalocyanine macrocycle11. However, the synthetic procedures and

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purification procedures of these complexes tend to be difficult12-13. We have discovered a rather
simple and elegant way to synthesize ruthenium-phthalocyanine compounds based on a template
method. The template-assisted approach involves the use of cis-[RuCl2(DMSO)4] as a precursor
that reacts with the appropriate dicyanobenzene to generate an intermediate with the nitrile ligand
chelating the ruthenium ion.
The cis-[RuCl2(DMSO)4] starting material was synthesized according to a previously
published procedure14. Reaction of this complex with 1.1 equivalents of 4-(3-carboxylic-
benzyloxy)-phthalonitrile (DCBz) (named here dicyanobenzene-R) in ethanol under reflux for 3 h
afforded cis-[RuCl2(DMSO)2(DCBz)], which was isolated in 76.0 % yield. The resulting structure
of dicyanobenzene compound will drive the reaction to kinetically favorable monosubstituted
phthalocyanine. The DCBz was obtained following a published procedure15. Briefly, the reaction
of 4-nitrophthalonitrile with 4-hydroxybenzoic acid in dimethylsulfoxide for five days at 120 ºC
afforded the desired product as 85.0 % in yield. The DCBz compound was characterized by mass
spectrum. The ESI-MS spectra were obtained using ultrOTOFQ-ESI-TOF Mass Spectrometer
(Bruker Daltonics, Billerica, MA, EUA). The electrospray solvent was 70:30 acetonitrile : milli Q
water. For each analysis, 10 L was introduced into the source at a flow rate of 5 L min-1. For
{DCBz}- we found m/z = 263.0449, consistent with the calculated isotopic patterns
1
(Supplementary material: Figure 1). Characterization by H NMR was also carried out.
Measurements were performed on a Bruker Avance III HD 400.15 MHz spectrometer in DCCl3
and DMSO-d6 as solvents at room temperature. Essentially the NMR samples were prepared by
dissolving 5−15 mg of the compound in 0.5 mL of the appropriate solvent. The 1H NMR spectra
of DCBz were in good agreement with the proposed structure (Figure 1). The 1H NMR spectrum
showed signals for the aromatic hydrogens between 8.2 – 7.0 ppm. The proximity of ether as well

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the acceptor character of carboxylic substituents causes downfield shifts in the plane of aromatic
ring for H-9 and H-13. A similar effect was observed for H-4 and H-5, which experience the effect
of the cyano group differently (depending on its position on the aromatic ring). A singlet signal at
13.04 ppm was observed and was assigned to the carboxylic acid substituent.
Figure 1: 1H NMR spectrum of 4-(3-carboxylic-benzyloxy)-phthalonitrile (DCBz) recorded in
DMSO-d6. δ(ppm): 7.25 (t, 1H, J1=2.7 Hz); 7.27 (t, 1H, J1=2.7 Hz); 7.54 (dd, 2H, J1=8.7 Hz, J2
= 2.6 Hz); 7.94 (d, 1H, J1=2.5 Hz); 8.02 (t, 1H, J1=2.7 Hz); 8.04 (t, 1H, J1=2.7 Hz); 8.15 (d, 1H,
J1 = 8.8 Hz); 13.04 (s, 1H).
The isolated [RuCl2(DMSO)2(DCBz)] was characterized by negative ion mode ESI-MS
and showed m/z = 592.9156 consistent with the proposed formula (Supplementary material: Figure
2). The mass spectrum also generated by ionization sputtering the [RuCl2(DMSO)2(DCBz)]

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complex to reveal the presence of a dimer forming a complex pattern suggesting fragmentation
followed by an electron transfer process (Scheme 1).
Scheme 1: The proposed fragmentation route of [RuCl2(DMSO)2(DCBz)] showing the formation
of some key fragment ions.
The monosubstituted phthalocyanine ruthenium complex [Ru(Pc-DCBz)] was obtained by
heating [RuCl2(DMSO)2(DCBz)] in a 5-fold excess of phthalonitrile at 130 ºC in dry pentanol
(Scheme 2). The crude product was obtained after 12 h at 0 C. It was purified by column
chromatography on basic silica eluting with tethrahydrofuran/toluene/dimethylformamide
(70:20:10). After evaporation on a rotary evaporator, the blue solid was dried in vacuum for 4 h to
give 54 % in yield based on the initial amount of [RuCl2(DMSO)2(DCBz)].

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Scheme 2: Template synthesis of [Ru(Pc-DCBz)] complex.
The [Ru(Pc-DCBz)] complex was characterized by mass, electronic absorption and FTIR
spectra. The electronic absorption spectra of [Ru(Pc-DCBz)] displayed a typical spectrum for
metallophthalocyanine compounds presenting intense π - π* absorption in the visible spectra. It
showed a band at 312 nm ( = 2.8 105 mol-1L-1), generally called the Soret or B-band, and a band
at 644 nm ( = 1.4 105 mol-1L-1), generally termed the Q-band, with a shoulder at 586 nm in
dichloromethane (DCM) solution. The [Ru(Pc-DCBz)] shows emission spectrum in DCM with

max = 690 nm after excitation at 640 nm. The FTIR spectrum was also similar to other ruthenium
phthalocyanine species such [Ru(Pc)] (Pc = phthalocyanine). The formation of [Ru(Pc-DCBz)]
was confirmed by the appearance of a carboxylic acid peak at 1670 cm-1, which was compared to
the FTIR spectrum of [Ru(Pc)] synthesized as described in a published procedure [15]
(Supplementary material: Table 1).
MALDI-TOF analysis of [Ru(Pc-DCBz)] with dihydroxybenzene (DHB) as a matrix, gave
mainly the two singly charged parent ions [Ru(Pc-DCBz)] (m/z = 749.89) and {[Ru(Pc-DCBz)]2}
(m/z = 1498.91). A third peak appeared with m/z = 1056.02 and was attributed to the association
of [Ru(Pc-DCBz)] with 2 DHB. Signals of weaker intensity corresponding to different fragment
ions were also observed (Supplementary material: Figure 3).

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Singlet oxygen generation (Δ1O2) of [Ru(Pc-DCBz)] and [Ru(Pc)] were determined in
DMSO using an earlier described procedure16. Briefly, the Φ1O2 values were calculated using 1,3-
diphenylisobenzofuran (DPBF) as a singlet oxygen scavenger. A solution of 2.0 x 10-6 M sensitizer
along with 2.5 x 10-5 M DPBF was irradiated using a 660 nm laser at 1 s time intervals from 0 to
10 s (Figure 2).
0
10
Figure 2: Changes in the absorption spectrum of DPBF in DMSO upon irradiation at 660 nm in
the presence of [Ru(Pc-DCBz)]. Recorded at every 1 s interval.
Singlet oxygen quantum yields were determined in air observed by the decrease in the
absorbance of DPBF monitored at 410 nm and compared with the decrease observed with the
standard photosensitizer, zinc phthalocyanine under identical conditions ( = 0.67 in DMSO17).
Values for the Φ1O2 of [Ru(Pc-DCBz)] was found 0.24 ± 0.03 and for [Ru(Pc)] was 0.35 ± 0.02
in DMSO. Cytotoxicity values of ruthenium-phthalocyanine compounds were measured by
determination of cellular proliferation using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. To B16F10 and MCF7 cell lines were added both

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ruthenium phthalocyanine sensitizers (1.0 x 10-5 M), incubated for 24 h, and irradiated at 660 nm
using a dose of 8.93 Jcm-2.
Figure 3 shows the cellular viability assays performed with [Ru(Pc-DCBz)] and [Ru(Pc)]
in murine B16F10 melanoma and human breast cancer MCF7 cells. Essentially there was no
cytotoxicity of either complexes in the studied cancer cell lines observed in the dark, although both
compounds were active under light irradiation at 660 nm. At 1.0 x 10-5 M concentrations both
[Ru(Pc-DCBz)] and [Ru(Pc)] yielded statistically the same value of the radiant exposure needed
to cause 50 % death of the treated B16F10 cells. However in MCF7 cells, the PDT activity of
[Ru(Pc-DCBz)] was higher than that of [Ru(Pc)]. The cell survival was found to be only 10 %
when [Ru(Pc-DCBz)] was used, while it was 85 % for the [Ru(Pc)] complex.
Figure 3: Cell viability plot of B16F10 and MCF-7 cancer cells treated with ruthenium
phthalocyanine compounds (10 M) for 24 h.
Moreover, [Ru(Pc-DCBz)] seemed to confer higher specificity and selectivity for breast
cancer cells, compared to [Ru(Pc)]. MCF-7 breast cancer cells are more differentiated (known to
be hormone responsive)18 compared to B16F10 melanoma cells. Confocal microscopy images
showed internalization of [Ru(Pc-DCBz)] in MCF-7 cells. A TRITC filter responding to red

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fluorescence signals was employed to recognize the location of [Ru(Pc-DCBz)] with red
fluorescence (Figure 4). The internalization is consistent with cell death process. That could be a
critical determinant for the cytotoxicity efficacy.
Therefore, subcellular localization is indeed more important than singlet oxygen quantum
yield in terms of overall cell killing19. New studies are underway to explore the cytotoxicity
pathways of these ruthenium-phthalocyanine compounds and will be presented shortly.
Figure 4: confocal laser scanning microscopy of MCF-7 breast cancer cells after incubation with
[Ru(Pc-DCBz)] for 1 h at 37 C (10 M).
Acknowledgments: This work was supported by CNPq, CAPES, FAPESP grant 2016/08851-0
and scholarship 2015/03746-9 (Laisa B. Negri). MRH was supported by US NIH grants
R01AI050875 and R21AI121700, Air Force Office of Scientific Research grant FA9550-13-1-
0068, by US Army Medical Research Acquisition Activity grant W81XWH-09-1-0514, and by
US Army Medical Research and Materiel Command grant W81XWH-13-2-0067.
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Design, synthesis and photobiological activity of novel ruthenium phthalocyanine complexes
Laisa B. Negri1, Tassia J. Martins2, Nicholas R. da Silva Gobo3, Kleber T. de Oliveira3*, Michael
R. Hamblin4,5* and Roberto S. da Silva1,2,4,5*
GRAPHYCAL ABSTRACT
The present work describes a novel and efficient synthetic method of ruthenium monosubstituted-
phthalocyanine complexes and its application against cancer cell lines. It allows find relationship of
subcellular localizations and cytotoxicity.

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Highlights
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-
-
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Synthesis of a ruthenium monosubstituted-phthalocyanine complex.
In vitro cytotoxicity assays on cancer cells show complexes possible tumoricidal role.
Confocal microscopy analysis on cancer cells was used to investigate cellular uptake.
Subcellular localization is dependent on phthalocyanine substituent.