Synthesis, Antileishmanial Activity and Spin Labeling EPR Studies of Novel β-Carboline-Oxazoline and β-Carboline-Dihydrooxazine Derivatives

A series of novel 1-(substituted-phenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8a-8i) and 1-(substituted-phenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9a-9h) derivatives, as well as their respective N-(chloroalkyl)-1-(substituted-phenyl)-9H-β-carboline-3-carboxamide precursors (6a-6i and 7a-7h), were synthesized and evaluated for their in vitro antileishmanial activity against promastigote and intracellular amastigote forms of Leishmania amazonensis. Compounds 8d, 8i, 9e and 9h exhibited significant activity for both promastigote and amastigote forms, with IC50 (50% inhibitory concentration) values ranging from 2.9 to 23.0 μM. In addition, spin label electron paramagnetic resonance (EPR) spectroscopy studies were carried out for the most active compounds against L. amazonensis promastigotes. The studies indicated that the tested compounds cause strong stiffness in the parasite plasma membrane and are capable of inducing internal metalloproteins oxidation of the parasite, resulting in their cross-linking to skeletal proteins. Compounds 8d and 8i produced the largest effect, showing that the presence of oxazoline group at C-3 of β-carboline nucleus is important for antileishmanial activity.


Introduction
Leishmaniases are a group of diseases caused by protozoan parasites from more than 20 Leishmania species that cause a variety of clinical manifestations in humans, among them, there are three main forms: visceral (VL), cutaneous (CL) and mucocutaneous. 1 The Leishmania amazonensis species, for example, is responsible for the anergic diffuse cutaneous form and the cutaneous forms with disseminated lesions. 2 According to World Health Organization (WHO), 1 97 countries and territories are endemic for leishmaniasis and it is estimated that between 600,000 to 1 million cases of CL occur worldwide annually. The current treatment for the leishmaniasis is mainly performed with pentavalent antimonials, amphotericin B, miltefosine and paromomycin. However, there is an increased incidence of treatment failure due to the toxicity and resistance exhibited by these drugs. 3 Besides this, no vaccines against Leishmania infections are available. 4 Therefore, it is of great importance to develop more active and less toxic compounds than the drugs used currently.

Synthesis, Antileishmanial Activity and Spin Labeling EPR Studies of Novel β-Carboline-Oxazoline and β-Carboline-Dihydrooxazine Derivatives
In this context, our research group has already demonstrated the antileishmanial activity of β-carbolines containing substituents at 1-and 3-positions of the β-carboline nucleus. [14][15][16][17][18][19][20] In the works developed by Tonin et al. 16 and Pedroso et al., 17 it was demonstrated the activity of N -alkyl-(1-phenylsubstitutedβ-carboline)-3-carboxamides against promastigote, axenic amastigote and intracellular amastigote forms of Leishmania amazonensis. The compound with the N-benzyl-carboxamide group at C-3 was active against promastigote and axenic amastigote forms with IC 50 (50% inhibitory concentration) values of 2.6 and 1.0 µM, respectively, 17 and killed L. amazonensis promastigotes through different cell death pathways, including apoptosis and autophagy. 18 Recently, the antileishmanial activity of β-carboline-1,3,5-triazine hybrids was reported by Baréa et al. 19 Among the compounds tested, the hybrid II ( Figure 1) showed potent activity against the promastigote (IC 50 = 6.2 ± 1.4 µM, selectivity index (SI) = 23.5) and amastigote (IC 50 = 1.2 ± 0.5 µM, SI = 121.4) forms of L. amazonensis and exhibited low toxicity. Studies of action mechanism in promastigotes showed that compound II caused alterations in cell division cycle and an increase of lipid-storage bodies, leading the cells to death through various factors. The accumulation of lipid bodies may be associated with apoptotic cell death. 19 Additionally, oxazoline and 5,6-dihydro-4H-1,3-oxazine heterocycles play an important role in organic synthesis, being present in the structure of various biologically active compounds. [20][21][22][23][24][25][26][27] For instance, the nitroimidazo-oxazole III ( Figure 1) showed IC 50 of 0.03 µM against the amastigote form of L. donovani DD8 transfected with luciferase, and was identified, from a series of 72 nitroimidazoles evaluated, as a candidate for the oral treatment of visceral leishmaniasis. This compound showed also in vivo activity in both rat and hamster models. 25 The dihydrooxazine phenylpyridine IV (Figure 1) was effective for L. donovani mouse model and L. infantum hamster model, displaying optimal efficacy, pharmacokinetic and safety, leading to its selection as a new candidate for treatment of VL. 26 Considering the promising antileishmanial properties of β-carboline nucleus, the synthetic and biological importance of oxazoline and 5,6-dihydro-4H-1,3-oxazine rings, and the need to develop antileishmanial agents more effective, in this work we designed new 1-(substituted-phenyl)β-carboline derivatives bearing oxazoline and 5,6-dihydro-4H-1,3-oxazine moieties at C-3 ( Figure 2). The novel 1,3-disubstituted-β-carboline derivatives were evaluated against promastigote and intracellular amastigote forms of L. amazonensis and their cytotoxicity were determined. The antileishmanial activity of N-(chloroalkyl)-β-carboline intermediates, precursors of the proposed derivatives, was also evaluated in order to verify the importance of the heterocyclic ring at the 3-position of β-carboline nucleus.
In addition, electron paramagnetic resonance (EPR) spectroscopy, associated with spin labeling method studies, was carried out for the most active compounds against L. amazonensis promastigotes. This analysis has been shown to be an important tool for analyzing the interaction of drugs or prototypes of drugs with parasite membranes. The literature describes the employment of this technique to evaluate the effects of miltefosine, 28,29 nerolidol 30,31 and parthenolide 32 on L. amazonensis membrane, and of elatol on Trypanosoma cruzi. 33

Antileishmanial activity
The β-carboline-oxazolines 8a-8i and β-carbolinedihydrooxazines 9a-9h were evaluated in vitro against the promastigote form of L. amazonensis ( Table 1). The compounds that showed IC 50 values greater than 100 µM were considered inactive. For the most active compounds against promastigotes, the antileishmanial activity for the intracellular amastigote form of L. amazonensis was also evaluated. The toxic effects on the host cells were determined by the selectivity index (SI). The SI for each active compound was calculated as the ratio between the cytotoxicity (CC 50 ) for macrophage J774-A1 cell lines and IC 50 against the promastigote and intracellular amastigote forms of L. amazonensis. (Table 1) for β-carbolineoxazolines 8a-8i shows that the presence of chlorine and dimethylamino substituents, at 2-and 4-positions of phenyl group linked to C-1, led to the active compounds 8d and 8i, respectively. Compound 8d showed also better selectivity indices (SI) for both forms of L. amazonensis than for the host cells (Table 1), being the most promising compound in this series.

Analysis of the IC 50 values
Concerning to 9a-9h series, most of β-carbolinedihydrooxazine derivatives showed moderate activity for L. amazonensis promastigotes, with IC 50 values ranging from 21.3 to 58.0 µM ( Table 1). The derivatives 9a, 9e and 9h containing the phenyl, 4-chlorophenyl and 4-methoxyphenyl substituents, respectively, at C-1 of β-carboline nucleus, were the most active compounds for promastigote form, exhibiting IC 50 values in the range of 21.3 to 27.5 µM, similar to that of reference drug miltefosine. 34 These compounds were then evaluated against intracellular amastigote form of L. amazonensis and showed IC 50 values in the range of 2.9 to 75.5 µM ( Table 1). The derivative 9e was the most promising, being 26 and 6 times more active than 9a and 9h, respectively. Besides that, 9e was 29.7 times more toxic for intracellular amastigotes than for macrophage J774-A1 cell lines, being a promising antileishmanial agent.

Spin label EPR spectroscopy studies
In order to investigate the interaction of the most active compounds for the promastigote form of L. amazonensis with the parasite membrane, EPR spectroscopy associated with the spin labeling method studies were carried out for 6d, 8d, 8i, 9a, 9e and 9h. Figure 4 shows the EPR spectra of the spin label 5-doxyl-stearic acid (5-DSA) incorporated in Leishmania membranes for samples untreated and treated with the studied compounds. EPR spectra showed that all compounds cause increases in parameter 2A // (outer hyperfine splitting) above the estimated experimental error (0.5 G), indicating decreases in molecular dynamics. In the treatment with 150 µM of compounds, some of them showed remarkable changes in the parasite membrane. Compounds 8d and 8i containing the oxazoline heterocycle at 3-position of β-carboline nucleus were the most effective for treatments at a concentration of 150 µM. However, we note that the compounds 6d, 9a, 9e and 9h can also cause high membrane stiffness at higher concentrations. In cell membrane the probe 5-DSA behaves as annular or boundary lipids that preferentially surround the hydrophobic surface of membrane proteins. 35 Because of these interactions with the transmembrane proteins, 5-DSA can monitor the dynamics at the periphery of proteins into the lipid bilayer. Thus, the changes in 5-DSA spectra caused by the compounds may be associated with changes in the membrane protein component.
Spin label EPR spectroscopy indicated that the treatments of L. amazonensis promastigotes with the studied compounds cause strong stiffness in the parasite plasma membrane. These strong membrane changes, with changes in parameter 2A // of ca. 5 G observed for two compounds at a relatively low concentration, cannot be explained by the simple presence of the molecules in the membrane, but must involve some oxidation process. Similar alterations in the EPR spectra of 5-DSA into plasma membrane were found in a previous study 36  has been shown that H 2 O 2 induces the formation of crosslinking of hemoglobin to skeletal proteins in the membranes of human erythrocytes in an azide phosphate buffer, associated with a progressive alteration of the cell's shape to echinocytic morphology, decreased cell deformability and increased phagocytosis. 37 Heme proteins were crucial for the occurrence of these cellular alterations, since they may be completely inhibited by previous exposure of red blood cells to carbon monoxide. Lipid peroxidation did not appear to be important because the antioxidant butylated hydroxytoluene decreased the fluorescent derivatives but did not prevent formation of the spectrin-Hb (hemoglobin) complex. 37 These observations suggest that the compounds tested are capable of inducing oxidation of internal metalloproteins of the parasite, resulting in their crosslinking to skeletal proteins.
Spin label EPR spectroscopy studies indicated that the tested compounds cause strong stiffness in the parasite plasma membrane and are capable of inducing internal metalloproteins oxidation of the parasite, resulting in their cross-linking to skeletal proteins. Compounds 8d and 8i produced the largest effect, showing that the presence of oxazoline group at C-3 of β-carboline nucleus is important for antileishmanial activity. Further studies will be conducted with these compounds aiming a better understanding of their mechanisms of action. Compounds 8d and 8i are also strong candidates for in vivo studies in view to the development of new antileishmanial agents.

General methods
All reagents were purchased from commercial suppliers, except the DMTMM that was synthesized according to the methodology described by Cronin et al. 38 and Kunishima et al. 39 The reactions were monitored by thin layer chromatography (TLC) conducted on Whatman TLC plates (silica gel 60 F 254 ). NMR spectra were recorded in a Varian spectrometer model Mercury plus BB at 300 (for 1 H) and 75 MHz (for 13 C) and in a Bruker spectrometer model Avance III HD at 500 (for 1 H) and 125 MHz (for 13 C), with deuterated solvents, chloroform (CDCl 3 ), methanol (CD 3 OD) and dimethyl sulfoxide (DMSO-d 6 ), and tetramethylsilane (TMS) as internal standard. Mass spectra (electrospray ionization mass spectrometry (ESI-MS)) were recorded on Thermoelectron Corporation Focus-DSQ II spectrometer. Melting points were determined in Microquímica apparatus model MQAPF-301 and are uncorrected. Spin label 5-DSA was purchased from Sigma-Aldrich (St. Louis, MO, USA).
The effects of compounds 6d, 8d, 8i, 9a, 9e a n d 9 h w e r e a l s o eva l u a t e d i n i n t r a c e l l u l a r amastigotes, in this antiproliferative assay, J774A1 macrophages (5 × 10 5 cells mL −1 ) and promastigotes (5 × 10 6 parasites mL −1 ) were added in a plate with coverslips and incubated at 34 °C with 5% CO 2 during 24 h. The treatment was performed after 24 h with compounds in increasing concentrations and incubated for 48 h. For the determination of IC 50 , the glass coverslips were fixed and stained with Panótico kit as indicated by the manufacturer and 200 macrophages per coverslip were evaluated on a light microscope. The number of macrophages infected, the number of amastigotes within each infected macrophage and the survival index (infected cells percentage × amastigote average per infected macrophage) were determined. Survival index of amastigotes from untreated infected macrophages was considered as 100% of survival.

Cytotoxicity assay
The cytotoxicity was evaluated in J774-A1 macrophages. The macrophages at concentration of 5 × 10 −5 cells mL −1 in RPMI 1640 medium supplemented with 10% FBS were introduced into sterile 96-well micro plates and incubated for 24 h at 37 °C and 5% of CO 2 tension. After this period, the supernatant was removed and increasing concentrations of the substances were added. After 48 h of incubation under the same conditions mentioned above, the cells were washed with 0.01 M PBS (phosphatebuffered saline) and 50 µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (2 mg mL −1 ) was added to each well and incubated at absence of light at 34 °C. After 4 h, 150 µL of DMSO was added in order to solubilize formazan crystals. The absorbance was read at 570 nm in microplate reader (Biotek Power Wave XS spectrofluorometer). The concentration that decreased 50% (CC 50 ) of viability of macrophages was determined by linear regression analysis of the data.

Statistical analysis
The data shown in the tables are expressed as the mean ± standard deviation of at least three independent experiments. The statistical analysis was performed using GraphPad Prism 6.0 software. 40 The samples were analyzed using one-way analysis of variance (ANOVA), and the Tukey post hoc test was used to compare means when appropriate. Values of p ≤ 0.05 were considered statistically significant.

Spin labeling and EPR spectroscopy
Promastigotes of L. amazonensis in suspension at 5 × 10 7 parasites mL −1 (2 mL) were incubated for 2 h at 26 °C in culture medium without fetal calf serum (FCS) and containing 150 µM of the treatment compound. After incubation, the sample was centrifuged at 1800 × g for 10 min to increase the cell concentration to 1 × 10 8 parasites mL −1 and decrease the final volume to 50 µL. To incorporate the spin label 5-DSA into the parasite membrane first a spin label film was made on the bottom of a glass tube. An aliquot (1 µL) of a 5-DSA ethanolic solution (4 mg mL −1 ) was added to the tube and after evaporation of ethanol the parasite suspension was placed on the film and stirred gently. Then, the sample was transferred to a 1-mm-i.d. (internal diameter) capillary tube for the EPR measurements.