Photodynamic effect of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin and (Zn2+ and Sn4+) derivatives against Leishmania spp in the promastigote stage: experimental and DFT study

Article abstract of DOI:10.1007/s11696-021-01702-y

In this work, we synthesized the meso-(aryl) porphyrin derivative 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP) and two meso-(aryl) metalloporphyrins, TCPP-Zn(II) and TCPP-Sn(IV), with reaction yields between 33.91 and 80.4%. The photophysical properties were determined, and the in vitro photodynamic properties were evaluated against Leishmania spp in the promastigote stage. The TCPP-Zn(II) reduced the viability of the parasites as shown by the lowest IC₅₀, which is even lower than the reference drug glucantime. In general, our results indicate that the beneficial effect related to the lethal capacity of porphyrins was greater in the presence of light irradiation. Furthermore, the inclusion of a diamagnetic metal such as Zn(II) is essential for the biological properties shown by this type of compounds, as it substantially improves their photodynamic activity. The Jablonsky diagrams were proposed using DFT computations, where the decay channels through the ground and excited states were assessed and the feasible generation of singlet oxygen was established, being TCPP-Zn(II) the one that shows a slightly more exothermic process than TCPP-Sn(IV).

Full text of DOI:10.1007/s11696-021-01702-y

Chemical Papers
https://doi.org/10.1007/s11696-021-01702-y
ORIGINAL PAPER
Photodynamic efect oꢀ 5,10,15,20‑tetrakis(4‑carboxyphenyl)
2
+
4+
porphyrin and (Zn and Sn ) derivatives against Leishmania spp
in the promastigote stage: experimental and DFT study
Fabián Espitia‑Almeida1,2,3 · Carlos Díaz‑Uribe · William Vallejo · Orlando Peña · Doris Gómez‑Camargo ·
1
1
1
2
4
5
6,7
Arnold R. Romero Bohórquez · Ximena Zarate · Eduardo Schott
Received: 4 February 2021 / Accepted: 13 May 2021
©
Institute of Chemistry, Slovak Academy of Sciences 2021
Abstract
In this work, we synthesized the meso-(aryl) porphyrin derivative 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP)
and two meso-(aryl) metalloporphyrins, TCPP-Zn(II) and TCPP-Sn(IV), with reaction yields between 33.91 and 80.4%.
The photophysical properties were determined, and the in vitro photodynamic properties were evaluated against Leishmania
spp in the promastigote stage. The TCPP-Zn(II) reduced the viability of the parasites as shown by the lowest IC , which is
5
0
even lower than the reference drug glucantime. In general, our results indicate that the beneꢀcial eꢁect related to the lethal
capacity of porphyrins was greater in the presence of light irradiation. Furthermore, the inclusion of a diamagnetic metal
such as Zn(II) is essential for the biological properties shown by this type of compounds, as it substantially improves their
photodynamic activity. The Jablonsky diagrams were proposed using DFT computations, where the decay channels through
the ground and excited states were assessed and the feasible generation of singlet oxygen was established, being TCPP-Zn(II)
the one that shows a slightly more exothermic process than TCPP-Sn(IV).
Keywords Photodynamic therapy · Porphyrin · Singlet oxygen · Leishmania spp. · Promastigote · DFT
Introduction
suꢁer from it around the world. Currently, leishmaniasis has
a wide distribution around the world, it is present in more
than 100 countries, and this disease is especially danger-
ous in developing and tropical countries (Alvar et al. 2012;
Gore and Nicholls 2012; Steverding 2017). Estimations
The study of leishmaniasis is a focus of vital interest for the
scientiꢀc and academic communities. Its importance of this
disease relies on the implied consequences for people who
5
*
*
William Vallejo
Instituto de Ciencias Químicas Aplicadas, Facultad de
williamvallejo@mail.unialtantico.edu.co
Ingeniería, Universidad Autónoma de Chile, Avenida Pedro
de Valdivia 425, Santiago, Chile
Ximena Zarate
6
ximena.zarate@uautonoma.cl
Departamento de Química Inorgánica, Facultad de Química
y de Farmacia, Centro de Investigación en Nanotecnología
Y Materiales Avanzados CIEN-UC, UC Energy Research
Center, Pontiꢀcia Universidad Católica de Chile, Vicuña
Mackenna, 4860 Santiago, Chile
1
2
Grupo de Investigación en Fotoquímica Y Fotobiología,
Facultad de Ciencias Básicas, Universidad del Atlántico, Cra
3
0 No 8-49, Puerto Colombia, Atlántico, Colombia
7
Grupo de Investigación UNIMOL, Facultad de
Medicina, Universidad de Cartagena, Cra. 6 No 36-100,
San Agustín, Cartagena, Colombia
Millenium Nuclei On Catalytic Processes Towards
Sustainable Chemistry (CSC), Concepción, Chile
3
4
Facultad de Ciencias Básicas Y Biomédicas, Universidad
Simón Bolívar, Cra 59 No 59-65, Barranquilla, Colombia
Grupo de Investigación en Compuestos Orgánicos de Interés
Medicinal (CODEIM), Parque Tecnológico Guatiguará,
Universidad Industrial de Santander, A.A. 678, Piedecuesta,
Colombia
Vol.:(0
1
123456789)
3

Chemical Papers
indicate that around 350 million people can suꢁer this dis-
ease, showing an annual incidence of 2 millions of people
and 30,000 deaths per year (Alvar et al. 2012; Khademva-
tan et al. 2017; WHO 2017). The leishmaniasis situation
becomes more serious since there are few pharmacological
alternatives available for the treatment of this disease. In
addition, the current therapeutic options in the market are
expensive, and besides, they involve several side eꢁects for
patients, including high toxicity, side eꢁects, resistance and
high cost (Cardona-Arias, et al. 2017; Corpas-López et al.
therapeutic alternatives. Last decade, photodynamic therapy
(PDT) has gained attention in cancer treatment, virus, bacte-
rial and parasites inactivation due to its versatility, eꢂciency
and less side eꢁect compared to traditional methodologies.
The PDT physical–chemical phenomena rely on the pho-
togeneration of reactive oxygen species (ROS) under elec-
tromagnetic irradiation on a photosensitizer. The generated
ROS are highly cytotoxic for diꢁerent biological substrates
(Lan et al. 2019; St Denis and Hamblin 2015; Yin et al.
2015). Porphyrins are versatile macrocycles; their photo-
physical properties are interesting to develop electronic and
medical applications (Marin et al. 2015). These compounds
have a series of remarkable physical and chemical proper-
ties (e.g., photostability, phototoxicity and singlet oxygen
production by irradiation with visible light). Furthermore,
these macromolecules are considered benchmarks in bio-
logical sciences as potential photosensitizing agents in anti-
cancer, antiviral, antifungal, antibacterial and antiparasitic
2
020; Glans et al. 2018). Table 1 lists some typical pharma-
cological treatments against Leishmaniasis.
Last decades, the pentavalent antimonial sodium sti-
bogluconate and meglumine antimoniate remain the most
important and cost-eꢁective antileishmanial drugs (Ong
et al. 2018). However, in recent years, Leishmania spp. has
demonstrated resistance against available therapeutic options
(
Olliaro et al. 2005), increasing the need of developing new
Table 1 Lists of some typical pharmacological treatments against Leishmaniasis (Rajora et al. 2017; Silva-Jardim et al. 2014)
Name/(reference)
Chemical structure
Administration
Side eꢁects
Meglumine antimoni-
ate (Glucantime®)
(15 mg/kg*day for
20 days)
High level of toxicity, arthral-
gia and myalgia
(
Araujo-Melo et al.
2
010; Silva-Jardim
et al. 2014)
Sodium stibogluco-
nate (Pentostam®)
(20 mg/kg*day for
10–20 days)
Hypotension, proteinuria,
myalgia, headache
(
Oliveira et al. 2011)
pentamidine -isethion-
ate (Balslev and
Nielsen 1992)
(3–4 mg/kg*day)
(AEP 2015)
bronchospasm, cough, head-
ache
Amphotericin B
10 mg/kg*day
Hyperpyrexia, renal tubular
damage, hepatitis
(
Laniado-Laborín
and Cabrales-Vargas
009; Silva-Jardim
et al. 2014)
2
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3

Chemical Papers
R
Besides, Neya et al. (1993) proposed a synthetic route
where aldehyde and pyrrole are mixed with propionic acid/
nitrobenzene or pyridine under reꢃux for 2–5 h. Porphyrin
synthesis under solvent-free conditions has reported interest-
ing results; Shy et al. reported fourfold acid-catalyzed con-
densation of aldehyde and pyrrole to yield a tetra-substituted
porphyrin through mechanochemical techniques (Shy et al.
2015). Among these methodologies, the Adler’s methodol-
ogy is a typical procedure used for porphyrin synthesis due
to the soft reaction conditions and high purity of the product.
The structural modiꢀcation in the meso-position of the
porphyrin is a common strategy to achieve target proper-
ties for PDT applications (Ormond and Freeman 2013a).
The β-substituted porphyrins containing amino-triazole or
amino-thiadiazole units have reported better singlet oxygen
production properties and higher values of relative aꢂnity
to the leishmanial arginase enzyme, improving the antipara-
sitic activity (Deda et al. 2020). Recently, Leszek reported
high singlet oxygen photogeneration properties in a series
of zinc porphyrins (monomer and dimer forms) (Mazur
et al. 2019). Ghazal et al. (2019) reported a singlet oxygen
R
N
N
CH CH COOH
3
2
NH
HN
R
N
Ref
30 min, 118 ºC
H
R
R
Scheme 1 Synthesis of meso-substituted porphyrins through Adler’s
reaction
1
. Condensation
H
R
R
H
H
R
N
H
TFA O BF -Et O
CHO
3
2
NH
HN
R
N
H
H
N
CH Cl , 25 ºC
2
2
H
R
2
.
Oxidation
R
H
R
R
R
R
R
H
R
N
H
N
quantum yield value of Φ = 0.87 for an asymmetrically
CH
Cl
, 25 ºC
Δ
2
2
NH
HN
NH
HN
substituted zinc phthalocyanine. There are other reports,
where the potential against leishmania parasite of other
sensitizers has been described. Linares et al. demonstrated
the antileishmanial eꢁect of three chlorin derivatives using
PDT (Linares et al. 2020). Metal-porphyrin derivatives have
shown to be eꢁective in reducing the viability of Leishma-
nia spp. (Gardner et al. 2010). In this sense, Andrade et al.
reported that under visible light irradiation the Zn(II) por-
phyrin meso-tetrakis (methylpyridinium-2-yl) reduced the
viability of L. brasiliensis more than 90%. Some reports
indicated that diamagnetic metallic porphyrin complexes
are characterized by relatively high excited triplet life-
times and singlet oxygen quantum yields, in contrast with
the paramagnetic metal porphyrin complexes (Guldi et al.
2000; Josefsen and Boyle 2008). In a recent work, our group
reported IC values of 1.2 µM against L. panamensis for
H
N
O
HO
N
H
CN
CN
Cl
Cl
R
Cl
Cl
H
CN
CN
O
OH
DDQH
DDQ
Scheme 2 Two-stage methodology proposed by Lindsey et al.
photodynamic therapy (Abada et al. 2012; Zoltan et al.
015). In this sense, medicinal chemistry makes use of these
2
characteristics to enhance a speciꢀc application, leading to
compounds with unique and suitable structural character-
istics for photodynamic therapy such as intense absorption
and electronic emission at wavelengths greater than 400 nm;
low energy HOMO–LUMO transitions; suitable redox and
photodynamic properties (Slomp et al. 2017; Yahia et al.
5
0
Zn(II) 5,10,15,20-Tetrakis(4-ethylphenyl)porphyrin deriva-
tive (Espitia-Almeida et al. 2020). In this work, we study the
eꢁect of meso-porphyrin and the Zn(II) and Sn(IV) deriva-
tives against Leishmania spp. in the promastigote stage. By
means of DFT calculations, it was possible to propose that
the Sn(IV) has a more exothermic decay pathway, which
would explain its lower photodynamic eꢂciency.
2
017). Some examples of porphyrin-type derivatives include
chlorins, benzoporphyrins and phthalocyanines (Kou et al.
017). Porphyrin-type macrocycles allow structural modi-
cations at the peripheral pyrrolic positions, and changes in
the synthetic methodologies can lead to new macrocycles
Gomes et al. 2018). The Adler’s methodology (Adler et al.
2
ꢀ
(
1
967) is a usual procedure used for the synthesis of porphy-
rins. This procedure consists of mixing equimolar amounts
of pyrrole and aldehyde under reꢃux in propionic or acetic
acid (Scheme 1).
Experimental
Synthesis and characterization
Other methodologies include the procedure proposed by
Lindsey et al. (1987). This synthesis of porphyrins occurs
in two stages (Scheme 2).
5,10,15,20-Tetrakis(4-carboxyphenyl)porphyrin (TCPP)
was synthesized according to Adler and collaborators
1
3

Chemical Papers
−
1
methodology (Adler et al. 1967), and the metallic-deriva-
tives TCPP-Zn(II) and TCPP-Sn(IV) were prepared accord-
ing to Manke and collaborators methodology (Manke et al.
(cm ): O–H (3150–3600), N–H (band overlapped by
1
O–H), C=O (1689), C=C (1609), C=N (1210); H NMR
(400 MHz, DMSO-d ) δ (ppm): 12.85 (4 H, s; O–H), 8.86 (8
6
2
014) (see scheme 3). Furthermore, we determined the
H, s, pyrrole-H), 8.22 (8 H, d; J=7.6 Hz, ortho-H), 7.85 (8
quantum ꢃuorescence yield (Φ ) and singlet oxygen quan-
H, d; J=7.66, meta-H), −2.98 (2 H, s, N–H). MS (ESI-IT),
f
1
+
tum yield (Φ ) for each compound. The H NMR spectra
m/z: 791.2 [M+H] . (Manke et al. 2014).
∆
were performed in a Bruker AC-400 spectrometer using
1
DMSO-d as solvent. The H chemical shifts are reported
5,10,15,20‑Tetrakis(4‑carboxyphenyl)porphyrini‑
6
in ppm (δ), relative to the residual signal of DMSO (signal
located at 2.5 ppm). The infrared spectrum was measured by
using an ECO-ATR alpha Bruker FTIR spectrometer. The
UV–Vis assay was recorded using a Shimadzu UV-2401PC
UV–Vis spectrophotometer; in this assay, we dissolved
nato‑Zicn(II), (TCPP‑Zn(II)) TCPP-Zn(II) was prepared
by mixing TCPP (0.5859 mmol) and ZnCl (1.4673 mmol)
2
in DMF (80 mL); the mixture was allowed to stir for 7 h at
100 °C open to the atmosphere (Manke et al. 2014). The
TCPP-Zn(II) was puriꢀed by means of column chroma-
tography using alumina as stationary phase and petroleum
ether:ethyl acetate (5:1) as the mobile phase (rf = 0.81).
Yield 0.402 g (80.4%), melting point > 300 °C; UV–Vis
(ethyl acetate) λ (nm): 422, 554, 592.
–
5
2
.0×10 g of each compound in ethyl acetate. Mass spec-
trometry assay was measured in ESI (LC)-MS/MS ion Trap
Amazon, Bruker spectrometer. The ꢃuorescence quantum
yield was determined by the comparative method using PTI
um 40 ꢃuorimeter. We determined melting point for using a
Plateau SNO10 melting point meter.
Dichloro‑5,10,15,20‑tetrakis(4‑carboxyphenyl)por‑
phyrinato‑Tin(IV) (TCPP‑Sn(IV) TCPP-Sn(IV) was syn-
thesized following a similar previously reported procedure
(Manke et al. 2014); TCPP (0.5514 mmol) and SnCl ·2H O
5
(
,10,15,20‑Tetrakis(4‑carboxyphenyl)porphyrin
TCPP) Synthesis of TCPP was carried out similar to the
literature procedures (Adler et al. 1967; Harrinam et al.
983; Ribó et al. 1994 cited in Manke et al. 2014) with slight
modiꢀcations; pyrrole (8 mmol) and 4-carboxybenzaldehyde
8 mmol) were mixed in propionic acid (100 mL); the mix-
2
2
(1.3297 mmol) were mixed in DMF (100 mL); this mixture
was left under stirring for 8 h at 100 °C open to the atmos-
phere. The TCPP-Sn(IV) was puriꢀed by means of column
chromatography using alumina as the stationary phase and
petroleum ether:ethyl acetate (10:1) as the mobile phase
(rf=0.60). Yield 0.263 g (52.6%); melting point >300 °C;
UV–Vis (ethyl acetate) λ (nm): 428, 557, 598.
1
(
ture was left under stirring for 10 h at room temperature
open to the atmosphere. The product (corresponding to the
TCPP) porphyrin was obtained by the addition of cool meth-
anol (80 mL) to the obtained mixture. Afterward, the TCPP
was puriꢀed by means of a column chromatography using
alumina as stationary phase and petroleum ether:ethyl ace-
tate 2:1 as mobile phase. The product showed an rf=0.80.
Yield 0.7596 g (33.91%); melting point>300 °C; UV–Vis
Photophysical study
We performed tests on the detection of reactive oxygen
species using sodium azide [the sodium azide is a speciꢀc
quencher of singlet oxygen (Bancirova 2011)] and noted
(
ethyl acetate) λ (nm): 416, 513, 548, 590, 644; FT-IR-ART
Scheme 3 Chemical synthesis
of TCPP and metal derivatives
TCPP-Zn(II) and TCPP-Sn(IV).
Into scheme: M=Zn(II), Sn(IV)
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Chemical Papers
a drastic reduction in the oxidation of DPBF. In a second
test, we added superoxide dismutase to the reaction medium
Biological study
[
the superoxide dismutase is a speciꢀc quencher of super-
Parasites culture
oxide radical (Bou-Abdallah et al. 2006)], and there was
no change in the oxidation of the DPBF; these results indi-
cate that the greatest contribution to the oxidation process
could be due to singlet oxygen. The singlet oxygen quantum
We used Leishmania panamensis and Leishmania brasil-
iensis for the in vitro study in their promastigote stages.
The parasites were cultured in RPMI-1640 supplemented
with 10% fetal bovine serum, 1% glutamine and 4% anti-
biotics (200U penicillin/200 μg Amikacin) under incuba-
yield (Φ ) of all studied compounds was determined open
Δ
to the atmosphere using the relative method with 1,3-diphe-
nylisobenzofuran (DPBF) as singlet oxygen trapping and
tion conditions 5% CO . The culture medium was renewed
2
5
,10,15,20-(tetraphenyl)-porphyrin (H TPP) as the refer-
every 48 h for 8 days, and the parasites were counted in a
Neubauer chamber (Guerra et al. 2016; Espitia-Almeida
et al. 2020).
2
ence standard (Akilov et al. 2007; Kiderlen and Kaye 1990;
Mosmann 1983; Taylor et al. 2010). Data for singlet oxygen
quantum yield calculation can be found in the supporting
information. Furthermore, the comparative method was used
to determine ꢃuorescence quantum yield, ꢃuorescein dis-
solved in sodium hydroxide (0.1 M) was used as standard,
and the sensitizers were dissolved in DMSO (Durmuş and
Nyokong 2008; Gomes et al. 2015; Guillaumot et al. 2016;
Khodov et al. 2015).
Cell viability
Cell viability was evaluated by the MTT method (Kid-
erlen and Kaye 1990; Moreira et al. 1996; Mosmann
1983; Ormond and Freeman 2013b; Wang et al. 2016a,
6
b); in summary: aliquots of 5 × 10 parasites were
exposed to five concentrations of each compound 1,
10, 50, 100 y 200 µM (dissolved in DMSO balanced
with 5% sodium phosphate buffer, pH = 7.4) for 24 h
in the presence of light and darkness (the irradiation
source was Omnilux lamps EL10000AG with a range
Computational details
To go deeper on the eꢁect of the metal on the macrocyclic
structures, both metalloporphyrins TCPP-Zn(II) and TCPP-
Sn(IV) were studied through computational simulations.
Density functional theory (DFT) and the time-dependent
approach (TDDFT) were used for the quantum chemical
computations. Optimizations of the molecular structures
of λ
= 420–450 nm for using light intensity
emission lamp
−
2)
80 J*cm , after 24 h of exposure, 50 µL MTT (5 mg/
mL) was added to the treatments, followed by 4 h of
incubation in the dark, and finally, the Optical Density
(OD) was measured. The results were graphed as per-
centage cell viability (%CV) vs compound concentration,
the mortality of parasites mediated by the decrease in
their %CV was directly attributed to the photoactivation
of the compounds, and thus, each experiment included a
negative control (parasite growth in the absence of com-
pounds), a positive control (reference drug: Glucantime)
and blank controls (Guerra et al. 2016; Espitia-Almeida
et al. 2020; Morgenthaler et al. 2008). All bioassay and
measurements of optical densities were taken in micro-
plates of 96 U-bottom wells, using the Multiskan Sky
ThermoScientific equipment. A total of 12 independ-
ent replicates of each test were performed—these were
correlated with a percentage variation coefficient < 5%.
Means and standard deviations were calculated, an
ANOVA test was applied to determine the differences
or similarities between treatments and the positive con-
trol, and finally, a post hoc analysis was performed using
Tukey statistics to check the statistical significance of
the results found, p < 0.05 (Espitia-Almeida et al. 2020;
Gomes et al. 2015).
of ground (S ) and excited states (singlets and triplets) of
0
these systems were performed with the functional Becke-
3
-parameter-Lee–Yang–Parr (B3LYP) (Becke 1993, 1998;
Lee et al. 1988) and the 6–31+G(d,p) basis set (Frisch et al.
1
984) for the non-metallic atoms and the LANL2DZ pseu-
dopotential (Wadt and Hay 1985) for Zn and Sn. Besides,
the long-range corrected hybrid CAM-B3LYP (Yanai et al.
2
004) and the Truhlar’s M06 functional which is a hybrid
meta-GGA (generalized gradient approximation) (Zhao and
Truhlar 2006, 2008) functionals were employed to compute
the vertical excitations. Harmonic vibrational frequencies
were computed to assess the structures as minima in the
potential energy surfaces. All calculations were performed
using the software Gaussian09 (Frisch et al. 2009). The per-
formed calculations at the optimized minima let us to obtain
the electronic properties and the corresponding adiabatic
excitation energies, which provided the proposed decay
route that the dyes might trigger to the photogeneration of
ROS, such as singlet oxygen.
The eꢁect of the solvent in the optimization procedures
was performed including ethyl acetate as solvent using a
dielectric constant of 5.9867, by means of the conductor-like
polarizable continuum model (CPCM) (Cossi 1998).
Because the porphyrins (TCPP, TCPP-Zn(II) y TCPP-
Sn(IV)) have poor solubility in the water, we chose ethyl
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Chemical Papers
acetate to carry out the photophysical characterization. The
biological testing was carried out using dimethyl sulfoxide
according to other reports (Morgenthaler et al. 2008; Pinto
et al. 2016; Abada et al. 2013). Furthermore, because of poor
solubility of the porphyrins in water, we did not extrapolate
our results to the aqueous systems.
Result and discussion
UV–vis study
Figure 1 shows the UV–Vis spectrum for three compounds
in ethyl acetate. TCPP shows the typical Soret band and the
four Q bands (Wang et al. 2016a). The electronic transi-
*
tion a (π)–e (π) is assigned to the Soret band. The bands
1
u
g
located at the range 513 nm and 644 nm are assigned to
*
Fig. 2 Emission spectra of TCPP, TCPP-Zn(II) and TCPP-Sn(IV). In
a (π –e (π) transitions (Lakowicz 2006). After metal
2
u
2u
g
DMSO at room temperature, pH=7.0
insertion, the TCPP-Zn(II) and TCPP-Sn(IV) spectra show
one Soret band and besides, a reduction in the Q bands num-
ber. After metallic insertion into porphyrin core, the symme-
try of porphyrin changes; the Q bands number decreases as
the metallic porphyrin has higher symmetry than porphyrin
results agree with the reported literature for the analogous
porphyrin and metalloporphyrin systems (Boscencu et al.
2012; Wang et al. 2016b). Furthermore, results show lower
ꢃuorescence emission intensity for TCPP-Zn(II) than TCPP,
which indicates a strong quenching of the porphyrin excited
singlet state (Vicente and Smith 2014). Table 2 lists the
photophysical properties for three compounds in DMSO.
(
Durmuş and Nyokong 2008).
Photophysical study
Figure 2 shows ꢃuorescence emission spectra for three com-
pounds in DMSO. Results showed that the Sn(IV) derivative
showed higher ꢃuorescent emission than TCPP metal-free
and TCPP-Zn(II). This might be due to the cross-linking
path, which is governed by orbital spin coupling in the
central atoms, so the ꢃuorescence properties of porphyrins
are based on the physicochemical properties of the central
atoms (Diaz-Uribe et al. 2014; Kadish et al. 2003). Our
The best result of Φ is obtained for TCPP-Zn(II). Previ-
Δ
ous reports indicated that diamagnetic metals such as Zn(II)
improve the production of singlet oxygen in porphyrin rings
and they are essential for the preparation of new organome-
tallic photosensitizing agents due to their easy incorporation
into the macrocycles; they generate high relaxation capacity
on the macrocycle system and allow the easy generation of
electronic transitions and spin splits of the electrons.
Computational study
Both porphyrins, TCPP-Zn(II) and TCPP-Sn(IV), were con-
sidered to assess the metal eꢁect on the measured optical
properties as well as the energy of their ground and excited
states, through quantum chemical simulations. First, the
theoretical characterization of the UV–Vis absorption bands
Table 2 Photophysical properties of compounds TCPP, TCPP-Zn(II)
and TCPP-Sn(IV)
Compound
λ
(nm)
λ
(nm)
max
Φf
ΦΔ
max
Absorption Fluores-
cence
TCPP
TCPP-Zn(II)
TCPP-Sn(IV) 428
416
422
651
606
650
0.23± 0.02 0.75± 0.04
0.10± 0.02 0.93± 0.04
0.35± 0.02 0.55± 0.04
Fig. 1 UV–Vis spectra of TCPP, TCPP-Zn(II) and TCPP-Sn(IV). In
ethyl acetate at room temperature, pH=7.0
1
3

Chemical Papers
was carried out by means of TD-DFT methods. These com-
putations provided the excited state of the vertical excita-
tions, their energies, oscillator strengths (f) and the active
molecular orbitals involved in the transitions. The results at
diꢁerent theoretical levels varying the functionals are shown
in Table 3. The reported excitations correspond to those with
the highest f, which show very good agreement with the
experimental energy values (nm) of the most intense bands
in the UV–Vis absorption proꢀles of the porphyrins, being
calculations carried out with B3LYP including implicit sol-
vation displayed a considerable improvement on the energy
values with respect to B3LYP without solvent eꢁect. In this
sense, we also obtained similar results using the Truhlar’s
M06 functional that includes 27% of HF exact electronic
exchange and corrections in the kinetic energy terms (see
Table 3). This functional has been reported as good approach
to simulate optical properties of porphyrin macrocycles
(Urzúa-Leiva et al. 2019; Zhao and Truhlar 2006, 2008).
On the other hand, we also performed calculations with the
long-range corrected functional CAM-B3LYP including
implicit solvation. With this functional, the values showed
more deviation than the other tested functionals, i.e., the
band of the TCPP-Zn appeared at 364 nm and for TCPP-Sn
at 375 nm.
4
22 and 428 nm for the Zn and Sn TCPP, respectively. The
Table 3 Energies and the oscillator strength (f) for TCPP-Zn(II)
and TCPP-Sn(IV) of the TDDFT computations to characterize the
absorption bands of the UV–Vis spectra
λ (nm)*
f
Active MO
%
The plots of these MOs isosurfaces are presented in
Fig. 3, and interesting results are observed. The MOs are
mostly localized along the porphyrin ring, with a contribu-
tion of chloride orbitals only in case of the HOMO of the
TCPP-Sn(IV) as shown in Fig. 3. Regarding the ROS pho-
togeneration, here we focused the theoretical calculations
to evaluate the possible routes to promote energy transfer
to triplet oxygen in DMSO, which would then generate sin-
glet oxygen. The found pathways are displayed in a scheme
shown in Fig. 4. The decay channels obtained for both por-
phyrins, TCPP-Zn(II) and TCPP-Sn(IV), display visible dif-
B3LYP
TCPP-Zn II
390
391
1.40 HOMO-1→LUMO
HOMO→LUMO+1
1.15 HOMO-1→LUMO+1
HOMO→LUMO
51
35
60
30
TCPP-Sn IV
B3LYP+Solv
TCPP-Zn II
407
406
1.69 HOMO-1→LUMO
HOMO→LUMO+1
1.57 HOMO-1→LUMO+1
HOMO→LUMO
56
35
62
33
TCPP-Sn IV
ferences. The adiabatic energies of both, the singlet (S ) and
1
M06+Solv
TCPP-Zn II
triplet (T ) states of the two systems, show diꢁerences of
1
406
406
1.87 HOMO-1→LUMO
HOMO→LUMO+1
1.66 HOMO-1→LUMO+1
HOMO→LUMO
56
41
60
38
~
0.1 eV. Speciꢀcally, the main diꢁerence is that for TCPP-
Zn(II), the S state undergoes intersystem crossing (ISC)
1
TCPP-Sn IV
to populate the triplet state T , which through fast internal
2
conversion (IC) process leads to the population of the adi-
The solvent implicit eꢁect was included via PCM using ethyl acetate
abatic T with energy 1.63 eV. Otherwise, the S state of
1
1
*
Experimental data correspond to 422 and 428 nm for TCPP-Zn II
TCPP-Sn(IV) undergoes ISC to populate a very close lying
and TCPP-Sn IV, respectively. The molecular orbitals (MOs) impli-
cated in the excitations are the frontier MOs, HOMO, HOMO-a,
LUMO and LUMO+1, where LUMO and LUMO+1 are degener-
ated in energy for both systems (using diꢁerent functionals)
state T following IC to T with energy 1.54 eV.
3
1
From the literature, it is known that ISC is a crucial pro-
cess to reach the triplet state of the photosensitizer for the
Fig. 3 Plots of the frontier
molecular orbitals of TCPP-
Zn(II) and TCPP-Sn(IV)
1
3

Chemical Papers
Fig. 4 Representation of the
proposed decay channel to
generate singlet oxygen through
energy transfer process. a
TCPP-Zn II and b TCPP-Sn IV
generation of singlet oxygen. So, our calculations propose
ion, in addition to improving the photodynamic properties,
improved the pharmacological eꢁect compared to the metal-
free compound TCPP and TCPP-Sn(IV) derivative. This
result is in concordance with other reports (Andrade et al.
2018). Furthermore, all compounds were more eꢁective to
decrease the cell viability of the L. brasiliensis and L. pana-
mensis at concentrations>50 µM in the irradiated assays.
These results showed a trend of intrinsic toxicity by the
three compounds, similar to that found by Morgenthaler
et al. 2008, this trend may also be attributed to the possible
activation of the compounds by residual portions of light
from the environment, because the compounds presented
3
that the excitation energy transfer to
⁠
O
⁠
occurring via
2
1
the described channels may produce ⁠ O . As it has been
2
⁠
reported, the process eꢂciency to reach singlet oxygen
depends on several facts such as thermodynamic aspects,
diꢁusion of the species, among others (Diaz-Uribe et al.
2
015; Díaz-Uribe et al. 2019). From them, it is worth high-
lighting that the energy available in the donor of the energy
transfer process should be higher than the
⁠
acceptor state of
1
O⁠ to go through an exothermic reaction. Thus, the herein
2
shown results and considering the adiabatic energy of the
optimized porphyrins in their T states, it is evidenced that
1
both porphyrins would be able to generate ROS, and TCPP-
high Φ , and thus, with small amounts of light the com-
Δ
Zn(II) shows the highest energy diꢁerence with respect
pounds could be activated and cause the death of the para-
1
to the reported experimental energy of
⁠
the O
⁠
(
⁠
1Δg) of
sites. Lethal concentrations 50 (IC ) were also determined
2
50
0
.97 eV (see Fig. 4).
for the 24-h treatments; these data are reported in Table 4.
The results show a signiꢀcant diꢁerence between the IC
5
0
Biology study
values toward L. brasiliensis under light irradiation and in
darkness (p < 0.05). The IC that was obtained for com-
5
0
Figure 5 shows the growth curves of the L. brasiliensis and
L. panamensis parasites in the promastigote stage exposed to
diꢁerent concentrations of TCPP, TCPP-Zn(II) and TCPP-
Sn(IV) under visible irradiation and in darkness. The com-
pounds were eꢁective in reducing parasite viability under
irradiated treatments (Fig. 5a, c). Additionally, an inverse
relationship between concentration and cell viability was
evidenced (Fig. 5a–d); thus, when the concentration of each
compound was increased during the treatments, the viability
of the parasites was reduced to a greater extent. The mortal-
ity of the parasites is directly attributed to the singlet oxygen
produced by the compounds TCPP, TCPP-Zn(II) and TCPP-
Sn(IV) when irradiated; in this sense the compound TCPP-
Zn(II) with lower Φ and higher Φ (Table 2) exerted high
pound TCPP-Zn(II) is lower than the control drug (Glucan-
time) against L. panamensis and L. brasiliensis (p<0.05),
as shown in Table 4, showing as a promising compound.
Therefore, we plan to continue our studies in the future per-
forming in vivo tests with mice.
Photochemical and photophysical studies on pho-
tosensitizers in photodynamic therapy relate the gen-
eration of singlet oxygen to biological activity (Akilov
et al. 2007; Docampo 1990; Morgenthaler et al. 2008).
In general, the photosensitizer acquires an excited con-
figuration, while oxygen in its triplet state is modified to
singlet oxygen, being the main pathway proposed for a
pharmacological response (Breitenbach et al. 2009; Hal-
liwell and Gutteridge 2015; Morgenthaler et al. 2008).
Based on the results obtained and the information found
in the literature, the following consideration regarding
the pharmacological mechanism of action can be sug-
gested; our hypothesis indicates that TCPP, TCPP-Zn(II)
and TCPP-Sn(IV), when irradiated with the light source,
generated reactive oxygen species (ROS), mainly singlet
f
Δ
antileishmanial activity against the parasites L. brasiliensis
and L. panamensis (p<0.05). Furthermore, the reduction in
viability in the parasites by the compounds in the dark was
not signiꢀcant (Fig. 5b, d), and the compound TCPP-Sn(IV)
with lower Φ exerted low leishmanicidal activity in the
Δ
irradiated tests. Therefore, the inclusion of the Zn(II) metal
1
3

Chemical Papers
Fig. 5 Phototoxic eꢁect of the
compounds TCPP, TCPP-Zn(II)
and TCPP-Sn(IV) against: a L.
brasiliensis under visible irradi-
ation (24 h), b L. brasiliensis at
darkness, c L. panamensis under
visible irradiation (24 h), d L.
panamensis at darkness. The
results plotted are the average
of 12 independent repetitions
of each test. Inside ꢀgure, Gluc
means (Glucantime)
Table 4 IC50 for TCPP,
Compound
Parasites
TCPP-Zn(II) and TCPP-Sn(IV)
against L. brasiliensis and L.
panamensis, 24-h treatments
IC50 (µM)± SD
L. brasiliensis
Light
L. panamensis
L. brasiliensis
L. panamensis
Darkness
TCPP
22.1± 0.8
7.1± 0.5
110± 2
16± 1
2.2± 0.4
86.1± 1
137± 1
103± 2
142± 3
120± 1
112± 2
116± 3
TCPP-Zn(II)
TCPP-Sn(IV)
Positive control
Glucantime
36.9± 0.8
12.7± 0.8
10.3± 0.3
8.8± 0.3
The results in the table are presented including the standard deviation obtained from 12 independent repeti-
tions of each test
1
3

Chemical Papers
oxygen (demonstrated in stages 2.2 and 2.3). Singlet oxy-
gen could be causing substantial damage to parasites at
the cell membrane level and even irreparable damage to
vital proteins of the parasites; another option is related to
modifications that may have been generated over the DNA
which does not allow DNA replication, producing the sud-
den death of the parasites (Breitenbach et al. 2009; Halli-
well and Gutteridge 2015; Skovsen et al. 2005; Docampo
Acknowledgements Fabián Espitia-Almeida thanks to COLCIEN-
CIAS (The call 727 - 2015). W. Vallejo and C. Diaz-Uribe thank
to Universidad del Atlántico. FONDECYT 1180565, FONDECYT
1
201880, ANID/FONDAP/15110019, Millennium Science Initia-
tive of the Ministry of Economy, Development and Tourism—
Chile, grant Nuclei on Catalytic Processes towards Sustainable
Chemistry (CSC).
Declarations
1
990). This hypothesis is further supported by the evalu-
Conꢀlict oꢀ interest On behalf of all authors, the corresponding author
ation of the pharmacological response in the absence of
irradiation, which was substantially low compared to irra-
diated trials. This could support the mortality of parasites
induced by ROS, especially by singlet oxygen. Finally,
we studied the effect of light on the growth and viabil-
ity of parasites, and there was no detectable effect on
the development of parasites, for which we compared the
viability of parasites of negative control (parasite + cul-
ture medium) in the absence of light vs. negative control
states that there is no conꢃict of interest.
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