Synthesis and antileishmanial activity of naphthoquinone-based hybrids

Introduction: leishmaniasis is a disease caused by protozoa of the genus Leishmania and is considered endemic in 98 countries. Treatment with pentavalent antimonials has a high toxicity, which motivates the search for effective and less toxic drugs. αand β-lapachones have shown different biological activities, including antiprotozoa. In recent studies, the isonicotinoylhydrazone and phthalazinylhydrazone groups were considered innovative in the development of antileishmania drugs. Molecular hybridization is a strategy for the rational development of new prototypes, where the main compound is produced through the appropriate binding of pharmacophoric subunits. Aims: to synthesize four hybrids of αand β-lapachones, together with the isonicotinoylhydrazone and phthalazinylhydrazone groups and to determine the antileishmania activity against the promastigotic forms of L. amazonensis, L. infantum and L. major. Results: β-lapachone derivatives were more active against all tested leishmania species. βACIL (IC50 0.044μM) and βHDZ (IC50 0.023μM) showed 15-fold higher activity than amphotericin B. The high selectivity index exhibited by the compounds indicates greater safety for vertebrate host cells. Conclusion: the results of this work show that the hybrids βACIL and βHDZ are promising molecules for the development of new antileishmania drugs.

Pharmacological therapy with pentavalent antimonials (N-methylglucamine antimoniate) is the first choice treatment, and amphotericin B and pentamidine are the second choice [4]. And there are successful reports of the use of miltefosine in cases of antimonium-resistant protozoa [5,6]. However, the treatment of leishmaniasis is still a difficulty, considering that the available pharmacological therapies present limitations in terms of efficacy and safety, prolonging the treatment; therefore, a range of adverse reactions, the need for parenteral administration, in addition to the possible emergence of resistance, lead to low adherence to treatment by patients.
Several efforts search for bioactive natural compounds that can be used in the treatment of parasitic diseases [7]. Lapachol, α-and β-lapachones are promising natural naphthoquinones for Medicinal Chemistry due to their structural properties. Goulart et al. described the leishmanicidal activity of lapachol and some derivatives, showing the pharmacological potential of these substances [8]. In 2013, Guimarães et al. presented the activity of lapachol, α-and β-lapachones against the promastigote forms of four species of the genus Leishmania, ratifying the relevance of these naphthoquinones in the development of new leishmanicides [9].
In a study published this year, Souza et al. (2020) pointed out the relevance of the presence of isonicotinohydrazone and phthalazinylhydrazone nuclei in the structures of compounds for the antileishmanial activity. In this work, the authors synthesized five hydrazones and evaluated in vitro the activity against the promastigote form of L. amazonensis [10].
The development of new substances with therapeutic potential is a complex task involving multi and interdisciplinary efforts. Several strategies are used by Medicinal Chemistry uses to make the process of developing new drugs more effective. Molecular planning is one of the crucial steps in the process, and Molecular Hybridization (MH) is an effective alternative for the rational design of molecular structures of new prototype compounds. In this proposal, hybrid compounds are the result of joining molecular structures of distinct bioactive compounds [11,12].  Considering the activity against protozoa of the genus Leishmania presented by the α-, β-lapachones and compounds containing the isonicotinoylhydrazone (ACIL) and phthalazinylhydrazone (HDZ) nuclei, four compounds were designer through the of MH strategy employing the molecular structures of the naphthoquinones and the ACIL and HDZ nuclei, as shown in figure 1. Subsequently, the hybrids were prepared and had their activities evaluated against the promastigote forms of L. amazonensis, L. infantum and L. major, besides the cytotoxic evaluation in murine macrophages.

Materials and methods
For the synthesis, all the reagents used were obtained from commercial sources and used without prior purification, with the exception of hydralazine hydrochloride, which was obtained from Apresolina ® pills (Anovis Industrial Farmacêutica Ltda) [10,13]. The synthesized substances had its melting temperatures determined in triplicate using analog fusiometer, model PFM-II (MS Tecnopon ® instrumentation). The chromatographic profile of the substances was determined by Analytical Thin Layer Chromatography (TLC), using 2x4 cm aluminum/silica gel 60 plates with UV 254 fluorescence indicator, revealed in ultraviolet (UV) or with iodine (I 2 ). The purification in chromatographic column used silica gel 60 (70 -230 mesh) and ethyl acetate/hexane (AcOEt/Hex) mixture with increasing polarity, as a mobile phase. After preparation and purification all, the synthesis products were stored under refrigeration and protected from light.
The structural elucidation of naphthhydrazones αACIL (1), αHDZ (2), βACIL (3) e βHDZ (4) was performed using 1 H and 13 C Nuclear Magnetic Resonance techniques ( 1 H and 13 C NMR) [13]. The NMR spectra have been registered in a Bruker Ascend TM 400 device, which operates at 400 MHz for the 1 H nuclei and at 100 MHz for the 13 C nuclei. The chemical displacements (δ) were given in ppm using tetramethylsilane solvent (TMS) as internal standard. All samples were solubilized in deuterated solvent (CDCl 3 or DMSO-d 6 ).

Extraction, purification, and characterization of lapachol
Lapachol was extracted from the stalk of the ipe (Tabebuia sp.). The splinters of the heartwood were submerged in an aqueous solution of sodium hydroxide (NaOH) at 1 % (m·v -1 ) for about 24 h. The filtrate was then acidified with a solution hydrochloric acid 6 M (HCl), and the lapachol was precipitated in the aqueous medium as a yellow solid. The solid was filtered by vacuum filtration and dried at room temperature.

Synthesis, purification, and characterization of β-lapachone
Cooled concentrated sulfuric acid (H 2 SO 4 ) (730 µL) was added to a reaction vessel (25 mL) containing lapachol (1 mmol; 242 mg) and immersed in an ice bath at 0 ºC. The reaction was stirred for 20 min. Then, the reaction mixture was poured into a beaker containing cold distilled water, the precipitated orange solid was filtered and dried at room temperature. b-lapachone was purified by column chromatography. General procedure for the preparation of the compounds αACIL (1) and βACIL (3) To a methanolic solution (5 mL) containing isoniazid (90 mg, 0.6 mmol) and the appropriate naphthoquinone (121 mg, 0.5 mmol) (α-or β-lapachone) was added a drop of concentrated HCl (37 %). The reaction mixture was stirred until complete consumption of naphthoquinone. The crystals formed were filtered, washed with methanol and distilled water and dried at room temperature. The product was purified by column chromatography [13].
General procedure for the preparation of compounds αHDZ (2) and βHDZ (4) In a solution containing hydralazine hydrochloride (1.5 mmol) in methanol (11 mL), H 2 SO 4 concentrate (approximately 700 µL) was slowly added, followed by appropriate naphthoquinone (121 mg, 0.5 mmol) (α-or β-lapachone). After the end of the reaction, the mixture was neutralized with 5 % (m·v -1 ) NaHCO 3 solution. The precipitate formed was filtered, washed with distilled water and dried at room temperature. The product was purified by column chromatography [13].

Determination of the mean inhibitory concentration (IC 50 ) of naphthhydrazones on the promastigotic forms of Leishmania amazonensis, Leishmania major and Leishmania infantum
The promastigote forms of Leishmania were kept cryopreserved in liquid nitrogen and in Schneider's Ò medium (Sigma, Chemical -USA) supplemented with Fetal Bovine Serum (FBS), 100 IU·mL -1 penicillin-streptomycin (Sigma) and glycerol as cryopreservative. To perform the assays, the parasites were thawed and kept in the same medium, without cryopreserver, at 26 ± 1 °C in a biological oxygen demand oven (Eletrolab EL202, São Paulo, Brazil). The promastigotic forms of Leishmania amazonensis (IFLA/BR/67/PH8), Leishmania major (MHOM/IL/80/Friendli) and Leishmania infantum (MHOM/5745) in stationary growth phase were washed in 0.9 % sterile saline solution, counted in Neubauer chamber and the volume adjusted to the desired concentration. The substances αACIL (1), αHDZ (2), βACIL (3) and βHDZ (4) were added to the microplate wells for cell culture, in triplicate, and serial dilutions were performed, reaching twelve concentration ranges (0.0097 to 20 µg·mL -1 ). Soon after, the parasites were sown in the amount of 1×10 6 leishmanias/100 µL of supplemented medium. The plate was then incubated in an oven at a temperature of 26 ºC for 48h and, 6h remaining for the end of this period, 20 µL of 1×10 -3 mol·L -1 resazurin was added, and the plate was incubated again. After the incubation period, the reading was performed on a 550 nm wavelength absorption plate reader (Biosystems model ELx800, Curitiba, PR, Brazil). For positive control, amphotericin B was used at a concentration of 2 μg·mL -1 , diluted in a supplemented Schneider's medium. The negative control was equivalent to Schneider's medium containing 1×10 6 promastigotes per well and, in this case, the viability was 100 % for the parasite. The reading of white, for each concentration and for the controls was necessary to disregard the absorbance resulting from the medium itself with interference or not of the substances studied. From these absorbances the concentration able to inhibit the growth in 50 % of the parasites was calculated (IC 50 ) [14,15].

Determination of the mean cytotoxic concentration (CC 50 ) of synthetic naphthohydrazone on RAW macrophages
The assessment of macrophage cytotoxicity was performed using the MTT assay (3-(4.5-dimethyl-2-thiazolyl)-2.5-diphenyl-2H-tetrazolium bromide). In the plates of 96 wells, 2×10 5 macrophages of the RAW 264.7 strain were incubated per well in 100 μL of RPMI 1640 medium (supplemented with 10 % SFB, 10 000 IU penicillin and 1000 IU streptomycin) in an oven at 37 °C and 5 % CO 2 for 4 h for cell adhesion. The supernatant was then withdrawn to remove the non-adhered cells. The DMSO solubilized naphthahydrazonic derivatives were diluted in RPMI supplemented medium, added to the plate containing the macrophages in serial concentrations reaching twelve ranges of final concentrations, starting from 100 μg·mL -1 , and incubated at 37 ºC and 5 % of CO 2 for 48 h. After this period, the cytotoxicity was evaluated by adding 10 % MTT at a concentration of 5 mg·mL -1 , diluted in 100 μL of RPMI medium, and the plate was incubated again for 4 h at 37 ºC and 5 % of CO 2 . At the end of this period, the supernatant was discarded and the formazan crystals were dissolved by adding 100 μL DMSO. Finally, the absorbance (550 nm) was measured using a Biotek plate reader (ELx800) [16]. The mean cytotoxic concentration CC 50 (µM) was determined from the linear portion of the curve, calculating the concentration of the compound that reduced the absorbance in treated macrophages by 50 % compared to negative control cells. The selectivity index (SI) was calculated by the ratio between CC 50 and IC 50 [17].

Statistical analysis
All the biological trials were performed in three independent experiments. The mean inhibitory concentration (IC 50 ) and the mean cytotoxic concentration (CC 50 ) with 95 % confidence limit were calculated using probit regression. Analysis of variance ANOVA followed by Bonferroni's test was performed taking p < 0.05 as the maximum level of statistical significance.

Antileishmanial activity of naphthohydrazones
The performance of in vitro tests of antileishmanial activity only in promastigote forms has been used as a screening test [16][17][18][19][20]. The in vitro evaluation of the antileishmanial activity of naphthohydrazones αACIL (1), αHDZ (2), βACIL (3) and βHDZ (4) has shown that these four hybrids were effective against promastigotic forms of L. amazonesis, L. infantum and L. major, this action being dependent of the concentration of the substance in test and the species of the parasite. When comparing the results obtained with the antileishmanial capacity of the precursor naphthoquinones (α-and β-lapachones) it was observed that the majority of the hybrids were more active.
The most active compounds against all the promastigotic forms of leishmanias tested were those that have the framework of phenanthrene, as occurs in β-lapachone and its hybrids βACIL (3) and βHDZ (4). While 3 had the lowest IC 50 (0.318 µM) against L. infantum, compound 4 was the most potent against L. amazonesis, with a IC 50 of 0.023 µM. Both hybrids, 3 and 4, were more active than the precursor compound, β-lapachone, on all tested strains of leishmanias. βACIL (3) was 65 times more active than β-lapachone (IC 50 2.90 µM in 72 h of experiment) [9] for L. amazonensis; whereas, βHDZ (4) was 126 times more active than the precursor orthonaphthoquinone in L. amazonensis. Compared to L. infantum, hybrids 3 and 4 were 2 times more active than β-lapachone (table 1).
When comparing the IC 50 of the synthesized naphthohydrazones with the amphotericin B, it can be affirmed that after exposure to L. amazonensis and L. major, the majority of the naphthohydrazones presented activity superior to the positive control. Highlight to βHDZ (4) over L. amazonensis and βACIL (3) compared to L. major, both with action 15 times greater than amphotericin B. Regarding L. infantum, the compounds 3 and 4 presented activity similar to the standard drug (table 1). Leishmaniasis are intracellular macrophage parasites in vertebrate hosts; therefore, assessing toxicity to these cells is essential when planning a drug for the treatment of visceral or tegumentary leishmaniasis [23]. The factor used to measure this safety was the selectivity index (SI), which is calculated by the ratio between the Cytotoxic Concentration for 50 % of macrophages (CC 50 ) and IC 50 . The molecules that present SI > 1 and SI > 20 are classified, respectively, as good and high safety, and those of high safety are qualified for further studies in infected macrophages and in vivo models [24,25].
The tested naphthohydrazones showed good safety for use as antileishmanial, highlighting the hybrid βACIL (3), 52 times more selective for L. amazonensis and L. major than for the host cell. The compounds that have the HDZ nucleus, αHDZ (2) and βHDZ (4), also showed high safety for mammalian cells with SI of 42,956 and 24,179, respectively, when used on promastigotes of L. amazonensis. These results qualify the naphthohydrazones αHDZ (2), βACIL (3) and βHDZ (4) for the trials on infected macrophages and in vivo models. Among the compounds tested, αACIL (1) and βHDZ (4) were the most selective for L. infantum in relation to macrophages, presenting SI of 8.821 and 8.043, respectively.
Although the mechanism of action of naphthohydrazones on leishmanias is not known, the results of this work show that the compounds promoted damage in the morphological structure of these parasites ( figure 3). Among these changes, the variation in parasite size, scourge and shape stand out. According to Rodrigues et al. (2014) [26] and Gadelha et al. (2013) [27], such changes may be caused by the destabilization of the tubulin-dependent cytoskeleton, since both the parasite's body shape and the scourge's integrity are highly dependent on the stability of the microtubules.
Although naphthohydrazones have relatively complex molecular structures, they were prepared using accessible compounds, the lapachol, a natural product from a renewable source; and two low-cost drugs, with known toxicity and pharmacokinetics, the isoniazid and hydralazine. In addition, classic reactions in mild conditions were used, and accessible synthetic routes are desired by the pharmaceutical industry, especially when it comes to drugs for the treatment of neglected diseases, such as leishmaniasis.
The promising antileishmania activity presented by naphthohydrazones, in association with the synthetic advantages of the route used, make the compounds αHDZ (2), βACIL (3) and βHDZ (4) suitable for the later stages of drug development.