In vitro anti-leishmanial effect of metallic meso-substituted porphyrin derivatives against leishmania braziliensis and leishmania panamensis promastigotes properties

Article abstract of DOI:10.3390/molecules25081887

In this study, a family of porphyrins based on 5,10,15,20-Tetrakis(4-ethylphenyl)porphyrin (1, Ph) and six metallo-derivatives (Zn²⁺(2, Ph-Zn), Sn⁴⁺(3, Ph-Sn), Mn²⁺ (4, Ph-Mn), Ni²⁺ (5, Ph-Ni), Al³⁺ (6, Ph-Al), and V³⁺ (7, Ph-V)) were tested as photosensitizers for photodynamic therapy against Leishmania braziliensis and panamensis. The singlet oxygen quantum yield value (Φ_?) for (1–7) was measured using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen trapping agent and 5,10,15,20-(tetraphenyl)-porphyrin (H2TPP) as a reference standard; besides, parasite viability was estimated by the MTT assay. After metal insertion into the porphyrin core, the Φ_? increased from 0.76–0.90 and cell viability changed considerably. The Φ_? and metal type changed the cytotoxic activity. Finally, (2) showed both the highest Φ_? (0.90) and the best photodynamic activity against the parasites studied (IC₅₀ of 1.2 μM).

Full text of DOI:10.3390/molecules25081887

molecules
Article
In Vitro Anti-Leishmanial Effect of Metallic
Meso-Substituted Porphyrin Derivatives against
Leishmania braziliensis and Leishmania panamensis
Promastigotes Properties
Fabian Espitia-Almeida 1, Carlos Díaz-Uribe 1, William Vallejo 1,
and Arnold R. Romero Bohórquez 3
*
, Doris Gómez-Camargo 2
1
Grupo de Fotoquímica y Fotobiología, Universidad del Atlántico, Puerto Colombia 810007, Colombia;
fespitia@mail.uniatlantico.edu.co (F.E.-A.); carlosdiaz@mail.uniatlantico.edu.co (C.D.-U.)
Grupo de Investigación UNIMOL, Universidad de Cartagena, Cartagena 130001, Colombia;
dmtropical@unicartagena.edu.co
Grupo de Investigación CODEIM, Universidad Industrial de Santander, Bucaramanga 680002, Colombia;
arafrom@uis.edu.co
2
3
*
Correspondence: williamvallejo@mail.uniatlantico.edu.co; Tel.: +57-5-3599484
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: M. Salomé Rodríguez-Morgade and Soji Shimizu
Received: 23 March 2020; Accepted: 17 April 2020; Published: 19 April 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: In this study, a family of porphyrins based on 5,10,15,20-Tetrakis(4-ethylphenyl)porphyrin
, Ph) and six metallo-derivatives (Zn2+ , Ph-Zn), Sn4+ , Ph-Sn), Mn2+ , Ph-Mn), Ni2+
, Ph-Ni),
Al3+ , Ph-Al), and V3+
, Ph-V)) were tested as photosensitizers for photodynamic therapy against
Leishmania braziliensis and panamensis. The singlet oxygen quantum yield value ( ∆) for (
(1
(
2
(3
(
4
(5
(
6
(7
Φ
1–7)
was measured using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen trapping agent and
5,10,15,20-(tetraphenyl)-porphyrin (H2TPP) as a reference standard; besides, parasite viability was
estimated by the MTT assay. After metal insertion into the porphyrin core, the Φ∆ increased from
0.76–0.90 and cell viability changed considerably. The Φ∆ and metal type changed the cytotoxic
activity. Finally, (2) showed both the highest Φ∆ (0.90) and the best photodynamic activity against the
parasites studied (IC50 of 1.2 µM).
Keywords: porphyrin; metalloporphyrins; photodynamic therapy; Leishmania braziliensis; Leishmania
panamensis; singlet oxygen
1. Introduction
Leishmania spp are extra and intracellular protozoan parasites that infect a variety of animals
(e.g., dogs, rodents, reptiles). However, this zoonosis also affects human beings when they invade the
habitat of both natural reservoirs and transmitting vectors thereof [
1,
2]. The parasite vectors are female
hematophagous mosquitoes of the genera Phlebotomus and Lutzomyia [3,4
]. The appearance of this
disease in humans can be observed on the skin surface, in mucous membranes, and in some organs (liver
and vessels). Among these, the cutaneous is the most frequent form of appearance. Those three clinical
presentations have distribution in more than 100 countries in five continents [
of 350 million people at risk of suffering from it. Currently, there are 12 million people infected,
with an annual incidence of 2 million people, with around 65,000 deaths reported per year [ ].
4], with an estimate
1,2,5,6
This disease is considered a priority problem for public health around the world, with greater interest
in poorest countries with high levels of malnutrition, economic and social inequality once they face the
biggest impacts and incidence [7,8]. Currently, the pharmacological treatments against this disease
(e.g., glucantime, miltefosine, pentamidine, isethionate, amphotericin B) have shown some degree of
Molecules 2020, 25, 1887; doi:10.3390/molecules25081887
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1887
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effectiveness against the parasites. However, due to their high toxicity and a wide range of adverse
effects, such treatments are controlled and restricted [ 16]. Besides, given the resistance observed
9
–
in recent years by Leishmania spp against all these therapeutic options [17], it is of utmost importance
to search for new therapeutic alternatives that are more effective, less toxic, and both safer and more
affordable for all vulnerable populations [18]. In this sense, due to their biological and photodynamic
properties to produce reactive oxygen species (ROS), especially singlet oxygen when they are irradiated
with visible light, porphyrin and metalloporphyrin derivatives have been used as an alternative tool in
photodynamic therapy (PDT) against Leishmania spp [19,20]. Previous studies reveal that metals affect
the stability of the porphyrin macrocycle and, therefore, metals can alter the photophysical properties
of the sensitizer; while enhanced intersystem crossing to the triplet state might be expected, followed by
metalation of porphyrins due to the heavy-atom effect. Complexes with diamagnetic metals (e.g., Zn)
have higher singlet oxygen quantum yields, since diamagnetic metals promote intersystem crossing
and have a long triplet lifetime [21–24]. This behavior is not reported for paramagnetic metals (e.g., Sn,
Al, V, Mn). In the case of substituents of intermediate size into the porphyrin core, the triplet lifetimes
are observed to decrease by up to two orders of magnitude. This is attributed to a distortion of the
macrocycle symmetry when the substituents “squeeze through” upon the hindered rotation of the
phenyl group [25,26]. Besides, amphiphilic groups can facilitate better delivery and accumulation of
porphyrins in the cells. Several studies have shown that the efficiency in photoactivity increases when
the number of carbon atoms in the side chains is increased [27]. The presence of a long alkyl chain was
shown to be important for high PDT efficiency of the amphiphilic tripyridyl porphyrins. Lesar et al.
showed that lipophilic moiety significantly improved PDT efficiency compared with the hydrophilic
analog which lacked the long alkyl chain [28]. Literature data suggest that hydrophilic porphyrins
linked to long hydrophobic chains are incorporated much easier into micelle formed by fatty substances.
According to expectations, the attachment of alkyl chain to the porphyrin molecule considerably
increased its hydrophobic properties [29]. Some reports suggest that when the substituent chain is
increased, a greater affinity of the sensitizer to the membranes is allowed, that is why its photodynamic
activity increases [30]. Ezzeddine et al. reported a progressive increase of lipophilicity from shorter
hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains which increased the phototoxicity of
the Zn(II) N-alkylpyridylporphyrins [31]. However, the photophysical properties, such as fluorescence
lifetime and quantum yield, of singlet oxygen do not change significantly [32
,33]. Furthermore,
β-substituted porphyrin systems have been evaluated against L. panamensis in the amastigote stage,
showing IC50 values between 5.7 and 24.1 µM [34]. Besides, regarding these compounds, there have
been reports of cellular viabilities <10% against L. major and L. braziliensis in the promastigote stage [35].
Other systems like the benzoporphyrins have shown suitable IC50 values (3.35 M) against L. major [36].
µ
Substantial improvements have been reported for these systems when metals are introduced into the
macrocycle core. Gomes et al. reported improvement of activity against L. amazonensis for inclusion
of Bi3+ and Sb5+, with IC50 values of 93.8
µ
M and 52.4
the cytotoxic activity of these systems against L. braziliensis in the promastigote stage was improved
after the inclusion of Zn2+
38]. In a recent publication, our group reported in detail the photophysical
and DFT results for ( ). In that study, we proposed that ( ) could be tested as sensitizers for
µM against L. amazonensis [37]. Moreover,
[
1–
7
1–7
photodynamic therapy [39]. In view of that, in the present study, our aim is to demonstrate the cytotoxic
activity of (1–7) against L. braziliensis and L. panamensis.
2. Results and Discussion
2.1. Singlet Oxygen Quantum Yield
The efficient interaction of the photosensitizer triplet state with the molecular oxygen ground state
may result in generation of singlet oxygen [40]. In order to determine
oxygen trapping agent and H2TPP as a reference standard. The generation of singlet oxygen by (
is evidenced by chemical trapping of singlet oxygen by DPBF, and the Φ∆ values of the compounds
Φ∆, DPBF was used as a singlet
1–7)

Molecules 2020, 25, 1887
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are listed in Table 1. The results indicate that (Ph-Zn, Ph-Mn, Ph-Al, Ph-V) had a quantum yield
higher than ( , Ph). This difference could be related to an increase of relaxation of excited states in
1
macromolecule; moreover, the insertion of these metals inside the ring generated more stability for the
generation of singlet oxygen [25,41,42].
Table 1. Singlet oxygen quantum yield (Φ∆) and fluorescence quantum yield (Φf) for (1–7).
Compound
ε (M−1cm−1 × 104)
Φf
Φ∆
1, Ph
5.0
2.5
6.0
4.0
7.0
3.4
3.1
0.11 ± 0.02
0.11 ± 0.03
0.76 ± 0.09
0.90 ± 0.03
0.76 ± 0.05
0.83 ± 0.03
0.68 ± 0.02
0.84 ± 0.04
0.86 ± 0.01
2, Ph-Zn
3, Ph-Sn
4, Ph-Mn
5, Ph-Ni
6, Ph-Al
7, Ph-V
0.32 ± 0.02
0.17 ± 0.02
0.08 ± 0.02
0.010 ± 0.005
0.0020 ± 0.0005
In general, the Φ∆ of (1–7) were lower for the paramagnetic metals than for the diamagnetic ones,
and this is in line with previous studies, which showed that porphyrins containing paramagnetic ions
were very poor photosensitizers [24 26]. It is possible that the introduction of low energy charge-transfer
,
states associated with disruption of the planarity of the macrocyclic ring system provides alternative
non-radiative deactivation channels. Finally, since Φ∆ values as low as 0.11 are known for porphyrins
derivatives in clinical trials, such as Lutetium Texaphyrin [43], and because singlet oxygen has been
implicated as an intermediary species leading to cell death following photoexcitation sensitizers agents
in photodynamic therapy [24], the results shown in Table 1 indicate that (
materials for photodynamic therapy.
1–7) are suitable as potential
2.2. Antileishmanicidal Activity
Several compounds have already used sensitizers against Leshmania species [44
,45]. However,
the search for new substances is an important topic in this research field. Compounds (
1–7) were
studied in the promastigotes stage of L. panamensis and L. braziliensis, with viability assessed by the
MTT assay. Figure 1 shows in detail the viability (%) results of L. braziliensis and L. panamensis with
incubation periods of 24 h in the presence of (
The results show that ( ) had the ability to effectively inhibit the parasites. In addition, a decrease in
the viability of the parasites was observed with the increase in the concentrations of the treatment.
Figure 1a,c show that, under light irradiation, the viability of ( ) was similar to the viability of the
Glucantime control for all ranges of concentration. These results are relevant, it verifies the potential of
the ( ) as sensitizers for PDT. Furthermore, the inhibitory activity was lower without light irradiation
for ( ), and this is due to the interaction of light with endogenous biomolecules [46]. When 200
of the compounds were used, ( , Ph-Zn) had the highest inhibitory activity against both L. braziliensis
1–7) both in the dark and under visible irradiation.
1–7
1–7
1–7
1
–7
µM
2
and L. panamensis, even the cell viability of (2, Ph-Zn) was the same as that of the Glucantime control.
According to Table 1, (Ph-Zn) had the highest Φ∆ (0.90), then under visible irradiation, the amount
of singlet oxygen available to attack the leishmania parasite is larger and the cytotoxic effect could be
bigger. The IC50 value (concentration that inhibited cell growth by 50%) was determined, and the
results are shown in Figure 2. In all cases of the tests, the activation of sensitizers by irradiation ensures
lower IC50 values. In the absence of light, the cytotoxic activity against the parasite was lower; the IC50
for (
are in line with other reports [20
under light irradiation, and (
parasites; only (Ph-Zn) had lower IC50 (1.2
(12.7 M) and in the dark (8.0 M) against L. panamensis. This result is associated with the biggest Φ∆
1
–7) was higher than 200
µ
M in the dark; all compounds required light activation—these results
47]. Compounds ( ) showed high toxicity against the parasites
) had IC50 similar of larger than the positive control against both
M) comparing to the positive control under irradiation
,
1–7
1, 3–7
µ
µ
µ

Molecules 2020, 25, 1887
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value of (
2
). Table 2 lists the IC50 values for compounds (
1–
7) under visible irradiation and without
irradiation (in the dark).
Figure 1. Parasite viability percentage results for compounds (
against L. braziliensis ( ) in the dark, ( ) under light irradiation; against L. panamensis (
(d) under light irradiation.
1
–
7
) with incubation periods of 24 h:
a
b
c) in the dark;
Figure 2. IC50 values for photoinactivation of L. braziliensis and L. panamensis promastigotes after 24 h
of incubation in the presence of (1–7) and the positive control under light irradiation.

Molecules 2020, 25, 1887
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Table 2. IC50 values for photoinactivation of L. braziliensis and L. panamensis promastigotes in the dark
and under visible irradiation.
L. braziliensis
L. panamensis
Compound
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
Under Irradiation
in the Dark
under Irradiation
in the Dark
1, Ph
2, Ph-Zn
3, Ph-Sn
4, Ph-Mn
5, Ph-Ni
6, Ph-Al
7, Ph-V
34.1 ± 1.8
11.6 ± 1.0
10.1 ± 0.7
59.0 ± 2.5
18.4 ± 2.5
50 ± 1.2
87 ± 3.5
–
117
>200
>200
>200
>200
>200
>200
10.3 ± 0.9
20.6 ± 1.3
1.2 ± 0.2
10.4 ± 0.8
50.0 ± 1.3
17.0 ± 1.0
15.6 ± 0.9
50.0 ± 2.1
–
105
>200
>200
>200
>200
>200
>200
8.8 ± 0.8
Glucantime
Besides, Figure 2 indicates that (
L. braziliensis. This result could be associated with the multi-resistance mechanism reported for
L. braziliensis [48 51]. The parasite inhibition mechanism is unknown and there is no clear report in
the literature [52]. However, after compounds ( ) were irradiated with visible light (see Table 1),
1–7) were more effective against L. panamensis than against
–
1–7
singlet oxygen was generated—this oxidant species could generate substantial damage to parasites
at the cellular membrane level and even irreparable damage to vital proteins or DNA that induce
death [52–54]. Our results suggest that singlet oxygen could be a reason for inactivation of the parasite.
It is clear that those compounds operate efficiently under visible light; in the dark the damage to the
parasites was not comparable to that of the positive control. Finally, these results are relevant and
show the potential of (1–7) as sensitizers for PDT, which indicate that (Ph-Zn) is the best candidate for
PDT applications.
3. Materials and Methods
3.1. Synthesis
All reagents were supplied by Aldrich. We synthesized porphyrin according to Alder and Cols
method [55], which relies on stirring aldehyde and pyrrole in propionic acid for 6 h at room temperature
and an oxygen atmosphere (see Scheme 1) [39]:
5,10,15,20-tetrakis(4-ethylphenyl)porphyrin (1): A mixture of pyrrole (8 mmol) and 4-ethylbenzaldehyde
(8 mmol) in of propionic acid (60 mL) was stirred by 6 h at room temperature in an open container.
The product was extracted from the reaction medium after addition of methanol (40 mL). We obtained
0.820 g of a bright purple powder that was purified through column chromatography using silica gel
(2.5
melting point > 300 ◦C; UV-Vis (ethyl acetate)
(3312.97), Csp3-H (2960.44), C=C (1685.54), C=N (1180.23), C-N (1020.47); 1 H RMN (400 MHz, CDCl3)
(ppm): 1.57 (12 H, t, J = 7.6 Hz, -CH2CH3), 3.02 (8 H, q, J = 7.6 Hz, -CH2CH3), 7.60 (8 H, d, J = 7.9 Hz,
3-HAr), 8.16 (8 H, d, J = 7.9 Hz, 4-HAr), 8.90 (8 H, s, Py); 13 C RMN (100 MHz, CDCl3)
(ppm): 15.56
4), 131.28 (Cβ-Py 8), 134.59
4); MS (ESI-IT), m/z: 727.2 [M + H]+; Anal. Elem. Calc.
×
24 cm) as stationary phase, and petroleum ether:ethyl acetate 5:1 (rf = 0.66). Yield: 0.680 g, 46%;
λ
(nm): 415, 512, 547, 590, 646; FT-IR-ATR (cm−1): N-H
δ
δ
(-CH2CH3
×
4), 28.96 (-CH2CH3
×
4), 120.21 (2-CAr
×
8), 126.23 (1-CAr
×
×
(3-CAr 8), 139.63 (Cα-Py
×
×
8), 143.62 (4-CAr
×
for C52H46N4 (%): C (85.91), H (6.39), N (7.71), Anal. Elem. Found. (%) C52H46N4, C (85.95), H (6.34),
N (7.71).

Molecules 2020, 25, 1887
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Scheme 1. Chemical synthesis of: (1) 5,10,15,20-Tetrakis(4-ethylphenyl)porphyrin and metal-derivatives,
(2) Zn (II), (3) Sn (IV), (4) Mn (III), (5) Ni (II), (6) Al (III), (7) V (III). Into scheme, X means Cl−.
Compound (
The mixture was stirred for 6 h at room temperature. Then, the reaction mixture was cooled in ice-water
bath; the formed precipitate was filtered and dried at room temperature; ( ) were purified through
column chromatography with silica gel (2.5 24 cm), petroleum ether:ethyl acetate (PE:EA) was used
2–7) were synthesized by mixing (1) with the metal chloride salt for each metal in DMF.
2–7
×
as mobile phase. Details of the spectroscopic characterization are listed in supplementary materials.
3.2. Singlet Oxygen Quantum Yield
The Φ∆ values of (
diphenylisobenzofuran (DPBF) as a singlet oxygen trapping agent and 5,10,15,20-(tetraphenyl)-
porphyrin (H2TPP) as a reference standard. The tests consisted of preparing a 1
10−9 M solution
1–7) were determined in air using the relative method with 1,3-
×
of each compound in Dimethylformamide (DMF) by triplicate, and calculations were determined
according to Equation (1) [56–59]:
W
Φ∆ = Φ∆st
×
(1)
Wst
where Φ∆st is the singlet oxygen quantum yield of standard H2TPP in DMF (0.64), W y Wst are the
DPBF photobleaching rates in the presence of complex (1 and 2) and standard porphyrin, respectively.
Data for Singlet Oxygen Quantum Yield calculation are provided in supplementary materials.
3.3. Fluorescence Quantum Yield
The comparative method was used to determine fluorescence quantum yield. Fluorescein
dissolved in water was standard, and sensitizers were dissolved in ethyl acetate. The fluorescence
quantum yield values were determined by taking the maximum of the Soret band as the excitation
wavelength (range 420–750 nm; slit = 2 nm). Quantum fluorescence yield was calculated with the
following equation [37,42,60,61]:
Fx ∗ Aest∗ n2est
Φx = Φfest
∗
(ec. 1)
(2)
Fest ∗ Ax ∗ n2x

Molecules 2020, 25, 1887
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where Fx and Fest correspond to the area under the curve in the fluorescence emission spectrum for
compounds (
for compounds (
1
), (
2
) and standard. Ax and Aest correspond to absorbance at excitation wavelength
), ( ) and standard; ïx and ïest correspond to the refraction index for solvents
1
2
(ïethyl acetate = 1.3724 and ïwater = 1.33336). Data for Singlet Oxygen Quantum Yield calculation are
provided in supplementary materials.
3.4. Parasites
Leishmania panamensis (M2903) and Leishmania braziliensis (UA140) were used in the in vitro
study. The parasites were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 1%
glutamine and 4% antibiotics (200 U penicillin/200 µg Amikacin) under incubation conditions of 5%
CO2. The metacyclic promastigotes in the infectious stage were isolated from stationary cultures of
5 days using a uniform procedure based on a modified density gradient purification.
3.5. Parasite Viability
Parasite viability was estimated by the MTT assay, converting a yellow tetrazolium salt,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into an insoluble product (formazan);
the amount of formed formazan depends on the number of viable parasites present [58,59,62].
The antileishmanicidal activity was studied at different concentrations in the presence and
absence of light. The irradiation source was Omnilux lamps (EL10000AG), with a range of
λemission lamp = 420 nm–450 nm for using light intensity 80 J
cm−2. All the measurements of the optical
·
densities were taken in microplates of 96 U-bottom wells, using the Multiskan Sky ThermoScientific
equipment. Standard deviation was obtained from 12 independent experiments—these were correlated
with a percentage variation coefficient <5%. We applied an ANOVA test to determine the differences or
similarities between treatments and the positive control. In addition, a post hoc analysis was performed
using Tukey statistics. Finally, differences were considered to be significant when p < 0.05.
4. Conclusions
Porphyrins (1–7) showed suitable singlet oxygen quantum yields, which induced inhibition of the
L. braziliensis and L. panamensis growth when the compounds were irradiated with a visible light source.
The non-irradiated treatments generated little or no inhibitory response of the parasites. All the results
indicate that (
showed better cytotoxic against L. panamensis than against L. braziliensis. Compound (
photosensitizer of all the compounds included in this study, as it showed a larger Φ∆ value (0.90)
1
–7) have suitable properties to be used in photodynamic therapy. All the compounds
2) was the best
and a better IC50 value compared to that of the positive control. Therefore, compound (2) is the best
candidate to be tested in photodynamic application against L. braziliensis.
Supplementary Materials: The following materials are available online, FTIR, Florescence, UV-Vis, singlet oxygen
plots data and the synthesis details.
Author Contributions: Conceptualization, C.D.-U., W.V., and F.E.-A.; methodology, C.D.-U., W.V., F.E.-A., D.G.-C.,
A.R.R.B.; software, F.E.-A., D.G.-C., A.R.R.B.; validation, C.D.-U., W.V., F.E.-A., D.G.-C., A.R.R.B.; formal analysis,
C.D.-U., W.V., F.E.-A., D.G.-C., A.R.R.B; investigation, C.D.-U., W.V., F.E.-A., D.G.-C., A.R.R.B.; resources, C.D.-U.,
W.V., F.E.-A., D.G.-C., A.R.R.B.; data curation, C.D.-U., W.V., F.E.-A.; writing—original draft preparation, C.D.-U.,
W.V., F.E.-A.; writing—review and editing, C.D.-U., W.V., F.E.-A., D.G.-C., A.R.R.B.; project administration, C.D.-U.,
W.V., and D.G.-C.; funding acquisition, C.D.-U., W.V., F.E.-A., D.G.-C. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by COLCIENCIAS for the grants. W. Vallejo and C. Diaz thank to Universidad
del Atlántico (RES. No 002047 – 10/12/2018. COD CB 22 TGI 2018).
Acknowledgments: F. Espitia would like to thank COLCIENCIAS (programa de apoyo Doctoral en la convocatoria
727-2015) for the grants. W. Vallejo and C. Diaz thank to Universidad del Atl
ántico (RES. No 002047 – 10/12/2018.
COD CB 22 TGI 2018).
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the freeze-dried powders are available from the authors.
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