In Vitro Anti-Leishmanial Effect of Metallic Meso-Substituted Porphyrin Derivatives against Leishmania braziliensis and Leishmania panamensis Promastigotes Properties.

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 (ΦΔ) 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 ΦΔ increased from 0.76–0.90 and cell viability changed considerably. The ΦΔ and metal type changed the cytotoxic activity. Finally, (2) showed both the highest ΦΔ (0.90) and the best photodynamic activity against the parasites studied (IC50 of 1.2 μM).


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 [4], with an estimate 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 [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

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 Φ ∆ , DPBF was used as a singlet oxygen trapping agent and H2TPP as a reference standard. The generation of singlet oxygen by (1-7) is evidenced by chemical trapping of singlet oxygen by DPBF, and the Φ ∆ values of the compounds  Table 1. The results indicate that (Ph-Zn, Ph-Mn, Ph-Al, Ph-V) had a quantum yield higher than (1, Ph). This difference could be related to an increase of relaxation of excited states in macromolecule; moreover, the insertion of these metals inside the ring generated more stability for the generation of singlet oxygen [25,41,42]. 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 (1-7) are suitable as potential materials for photodynamic therapy.

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 (1-7) both in the dark and under visible irradiation. The results show that (1-7) 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 (1-7) was similar to the viability of the Glucantime control for all ranges of concentration. These results are relevant, it verifies the potential of the (1-7) as sensitizers for PDT. Furthermore, the inhibitory activity was lower without light irradiation for (1-7), and this is due to the interaction of light with endogenous biomolecules [46]. When 200 µM of the compounds were used, (2, Ph-Zn) had the highest inhibitory activity against both L. braziliensis 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 IC 50 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 IC 50 values. In the absence of light, the cytotoxic activity against the parasite was lower; the IC 50 for (1-7) was higher than 200 µM in the dark; all compounds required light activation-these results are in line with other reports [20,47]. Compounds (1-7) showed high toxicity against the parasites under light irradiation, and (1, 3-7) had IC 50  value of (2). Table 2 lists the IC 50 values for compounds (1-7) under visible irradiation and without irradiation (in the dark).        Besides, Figure 2 indicates that (1-7) were more effective against L. panamensis than against L. braziliensis. This result could be associated with the multi-resistance mechanism reported for L. braziliensis [48][49][50][51]. The parasite inhibition mechanism is unknown and there is no clear report in the literature [52]. However, after compounds (1-7) were irradiated with visible light (see Table 1), 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][53][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.

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

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]: Compound (2-7) were synthesized by mixing (1) with the metal chloride salt for each metal in DMF. 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; (2-7) were purified through column chromatography with silica gel (2.5 × 24 cm), petroleum ether:ethyl acetate (PE:EA) was used as mobile phase. Details of the spectroscopic characterization are listed in supplementary materials.

Singlet Oxygen Quantum Yield
The Φ ∆ values of (1-7) were determined in air using the relative method with 1,3diphenylisobenzofuran (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 of each compound in Dimethylformamide (DMF) by triplicate, and calculations were determined according to Equation (1) [56][57][58][59]: where Φ ∆st is the singlet oxygen quantum yield of standard H 2 TPP in DMF (0.64), W y W st 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.

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]: where F x and F est correspond to the area under the curve in the fluorescence emission spectrum for compounds (1), (2) and standard. A x and A est correspond to absorbance at excitation wavelength for compounds (1), (2) and standard; ï x and ï est correspond to the refraction index for solvents (ï ethyl acetate = 1.3724 and ï water = 1.33336). Data for Singlet Oxygen Quantum Yield calculation are provided in supplementary materials.

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% CO 2 . 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.

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 λ emission 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.

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 (1-7) have suitable properties to be used in photodynamic therapy. All the compounds showed better cytotoxic against L. panamensis than against L. braziliensis. Compound (2) was the best photosensitizer of all the compounds included in this study, as it showed a larger Φ ∆ value (0.90) and a better IC 50 value compared to that of the positive control. Therefore, compound (2) is the best candidate to be tested in photodynamic application against L. braziliensis.