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In vitro Leishmanicidal and Trypanosomicidal Properties of Imidazole‐Containing Azine and Benzoazine Derivatives

Abstract Leishmaniasis and Chagas diseases are two of the most important parasitic diseases in the world. Both belong to the category of Neglected Tropical Diseases, and they cannot be prevented by vaccination. Their treatments are founded in outdated drugs that possess many pernicious side‐effects and they're not easy to administer. With the aim of discovering new compounds that could serve as anti‐trypanosomal drugs, an antiparasitic study of a synthetic compound family has been conducted. A series of new 1,4‐bis(alkylamino)‐ and 1‐alkylamino‐4‐chloroazine and benzoazine derivatives 1–4 containing imidazole rings have been synthesized and identified. Their structures showed a possible interest based on previous work. Their in vitro anti‐Leishmania infantum, anti‐L. braziliensis, anti‐L. donovani and anti‐T. cruzi activity were tested, as well as the inhibition of Fe‐SOD enzymes. It was found that some of them exhibited quite relevant values indicative of being worthy of future more detailed studies, as most of them showed activity to more than only one parasite species, especially compound 3  c was active for the three studied Leishmania species and also for T. cruzi, which is a very interesting trait as it covers a wide spectrum.

Leishmaniasis and Chagas diseases are two of the most important parasitic diseases in the world. Both belong to the category of Neglected Tropical Diseases, and they cannot be prevented by vaccination. Their treatments are founded in outdated drugs that possess many pernicious side-effects and they're not easy to administer. With the aim of discovering new compounds that could serve as anti-trypanosomal drugs, an antiparasitic study of a synthetic compound family has been conducted. A series of new 1,4-bis(alkylamino)-and 1alkylamino-4-chloroazine and benzoazine derivatives 1-4 containing imidazole rings have been synthesized and identified.
Their structures showed a possible interest based on previous work. Their in vitro anti-Leishmania infantum, anti-L. braziliensis, anti-L. donovani and anti-T. cruzi activity were tested, as well as the inhibition of Fe-SOD enzymes. It was found that some of them exhibited quite relevant values indicative of being worthy of future more detailed studies, as most of them showed activity to more than only one parasite species, especially compound 3 c was active for the three studied Leishmania species and also for T. cruzi, which is a very interesting trait as it covers a wide spectrum.
Leishmaniasis and Chagas disease count among the most extended tropical infections caused by protozoan parasites. Both diseases are globalized. [1] Trypanosoma cruzi is responsible for Chagas disease, that consists of two clinical phases: acute and chronic. In its chronic phase is responsible of severe cardiac and gastrointestinal injuries, [2] whereas more than twenty Leishmania species and subspecies are the cause of leishmaniasis, which encompasses a wide spectrum of diseases producing injuries at the cutaneous, mucocutaneous and visceral levels.
Visceral leishmaniasis is the most severe form among them, being responsible of thousands of deaths every year. It is caused by several subspecies included in the L. donovani complex. Cutaneous and mucocutaneous leishmaniasis are, however, more extended, and are prevalent in Latin America, where they are caused mainly by the L. mexicana, L. braziliensis, and L. panamensis species. [3] Chagas disease can also reactivate the parasitemia when the host underwent immunosuppression. Some patients who were thought to be cured suffered a reactivation when treated with anticancer chemotherapy, had a liver or kidney transplant, or were diagnosed with AIDS. This reactivation also occurred with meningoencephalitis and/or acute myocartitis. The infection persists in patients with chronic chagasic cardiopathy that have a heart transplant, only a temporary remission of parasitemia takes place when treated with benznidazole. [9][10][11] Due to the large number of people affected by these tropical infections every year, great efforts have been made to find and design therapeutic agents with leishmanicidal and/or antichagasic properties. Historically, two heterocyclic compounds (benznidazole and nifurtimox) have been broadly used against Chagas and both are useful in the treatment of the acute phase, but not so when the disease reaches the chronic stage, because of low effectiveness and undesirable side effects. [12][13][14] Many other drugs have been tested in recent times with a wide variety of results, [15] but none of them are as extended in the clinical practice as the aforementioned compounds, and extensive research remains in search of new effective, safe and easy to synthesize molecules.
A similar picture is obtained when considering the current anti-leishmania treatments. Pentavalent antimonials have been the matter of choice for many years, but resistance of the parasite against those drugs is now widespread in many countries, mainly in the Indian subcontinent. More recently, molecules like amphotericin B or miltefosine have shown great efficacy against leishmaniasis, but their syntheses are expensive, and their use is not exempt of therapeutic complications. [16,17] Therefore, the design of new molecules effective and without negative side-effects against those parasitic diseases remains as a goal for many research groups.
The research of novel agents against diseased caused by trypanosomatid is centered against certain targets, mainly in metabolic pathways or key survival parasitic enzymes. In recent years, cysteine proteinase, hypoxanthine-guanine phosphoribosyl transferase, trypanothione reductase, sterol metabolism, kinetoplast DNA sites and such have been the main studied targets. [18] The present work is focused on iron superoxide dismutase (Fe-SOD), due to its specificity, as the equivalent molecule found in mammals appears linked to copper or zinc atoms (Cu/ Zn-SOD). Malfunction of this enzyme compromises parasite survival greatly, as it is the parasite main defence against oxidative burst (and consequent generation of reactive oxygen species). [19] For this reason, this molecule is an interesting target for study, as the interaction of prosthetic groups with the active sites containing the metallic ion deactivate the enzyme and its antioxidant effect.
In connection with this matter, our group has been working in the last years in the synthesis and antiparasitic properties of different series of phthalazine derivatives functionalized with imidazole or pyrazole rings at the end of sidechains attached to the pyridazine moiety. [20][21][22][23][24] According to the antiparasitic activity reported previously for many azole compounds, some of these series containing the imidazole moiety have shown interesting activity data against T. cruzi and/or different Leishmania species, and those properties seem to be related to the simultaneous presence of both the pyridazine and the imidazole heterocyclic systems, clearly showing structure dependence for both trypanocidal and leishmanicidal features.
In previous work we found that the imidazole and pyrazole derivatives inhibited the Fe-SOD activity on T. cruzi, [21] and Leishmania sp. species, [23] while their impact on human CuZn-SOD was not significant.
In this work we ponder the effect of modifications in the structure of the azine moiety on the leishmanicidal and trypanosomicidal properties in several series of related structures. Four groups of compounds were tested in vitro against three Leishmania species: L. braziliensis, as a representative parasite among those causing mucocutaneous leishmaniasis, L. donovani as a representative agent for visceral Leishmaniasis and L. infantum as a representative of cutaneous Leishmaniasis, and also against T. cruzi. All compounds tested ( Figure 1) include the imidazole ring at the end of a sidechain linked to the exocyclic amino group but differ in the disposition of the nitrogen atoms located in the azine or benzoazine moiety, in the number of methylene groups conforming the sidechain, in the attachment point of the sidechain to imidazole or benzimidazole moieties or in the presence/absence of chlorine atoms attached to the azine ring.

Chemistry
The preparation of compounds 1 a-b, 1 e-f, 3 a-d and 4 a-d was performed in every case through nucleophilic substitution of the chlorine atom at the corresponding monochloroazine or monochlorobenzoazine by the required imidazole-containing aliphatic amine [20][21][22] (Scheme 1) while compounds 1 c-d were prepared in a similar way using 2,4-dichloroazine with only one chlorine atom substitution (Scheme 2). Xylene or dry ethanol were alternatively used as the solvent depending on the solubility features of reactants and products. In most cases, trimethylamine was added.
Compounds 2 a-d were prepared from 1,4-dichlorophthalazine according to the methodology shown in Scheme 3, following conditions related to those previously established by our group for the synthesis of similar compounds. [20][21][22] The mono-and bis(alkylamino)substituted derivatives 2 a and 2 b were obtained by nucleophilic substitution at the C-1 or simultaneously at C-1 and C-4 positions of the starting compound with 2-(1H-benzo[d]imidazol-2-yl)ethylamine at 100°C in a reactor, with dry ethanol as the solvent, for 24 hours, using triethylamine as the chlorine acceptor (method A). [21] The synthesis of the 2 c and 2 d was achieved in a similar way using 2-(imidazo[1,2-a]pyridin-2-yl)ethylamine by performing the reaction under reflux of toluene during 48 hours (method B). [24] Isolation of the compounds from the crude reaction mixtures was performed by column flash chromatography with a chloroform/methanol 8 : 2 mixture.
All of the newly synthesized compounds were unequivocally identified by their analytical, ESI mass spectra, IR, 1 H NMR, and 13 C NMR spectroscopic data, as shown in the Experimental Section. Heteronuclear multiple quantum coherence (gHMQC) experiments were performed for an accurate assignment of the 1 H and 13 C NMR spectra signals, obtaining especially useful results.
The mono-and bis(alkylamino)substitution products 2 a-d were easily differentiated in both 1 H and 13 C NMR spectra on the basis of signals corresponding to the ring atoms of the phthalazine system. [21] Protons H 5 and H 8 , lost their equivalence in the monosubstituted compounds 2 b and 2 d, as the H 8 proton, close to the alkylamine chain, was deshielded with respect to the proton neighbouring the chlorine atom (H 5 ). Accordingly, the carbon atom attached to the alkylamino substituent (C 1 ) was always deshielded with respect to the carbon neighbouring the chlorine atom in the 13 C NMR spectra.

In vitro leishmanicidal and trypanosomicidal evaluation
The in vitro activity, toxicity and selectivity index of compounds Although extracellular forms are more commonly used due to ease of working with them, they are less indicative of antiparasitic activity, since Leishmania extracellular forms are only transitory in vertebrate hosts and in any trypanosomatid infection, amastigotes last longer in the host and are responsible of cell destruction and thus, symptoms. [25] Therefore, intracellular assays were performed by infecting macrophage cells of the leishmanial species with promastigotes, which were transformed into amastigotes within 1 day of infection and then treated with the compounds. [24] Compounds 1-4 were also tested against extracellular epimastigote and axenic amastigote forms of T. cruzi SN3 strains. Finally, the cytotoxicity of compounds 1-4 was evaluated using J774.2 macrophages for Leishmania sp assays and mammalian Vero cells in T. cruzi cases as the cellular model. Selectivity indexes were calculated in all cases and results obtained for the reference drug used in each case were included for comparison. In order to get a picture of compounds effectiveness, the followed criteria were a IC 50 close to 10 μM and a Selectivity Index (SI) higher than 20 for Leishmania species and higher than 50 for T. cruzi. [26] Tested compounds were less cytotoxic than BZN in T. cruzi assays and most of the tested compounds were also less cytotoxic than Glucantime in Leishmania assays, being compounds 2 c and 2 d the only exception, although no clear correlation between differences in their structures and measured toxicity could be found.
Regarding the in vitro data, the more informative selectivity index (SI) values were calculated, being shown in Tables 1-4. When analysing the calculated number of times that the SI of each compound exceeded the SI of Glucantime or BZN (in brackets), very good results were found, mainly for compounds 3 c and 3 d, which achieved high values in all the amastigote forms. These values are shown in bold type in the Tables.
The specificity index (SPI) was calculated for results in Leishmania. Obtained results were between the range of 0.4 < SPI < 2, (data not shown) which indicate that the studied compounds were effective against both forms. [27] The following step in the activity study, alterations caused by the most active compounds among the eighteen synthesized on the infectivity was determined. J774.2 macrophage cells were then infected in vitro with promastigote forms of L. infantum, L. braziliensis or L. donovani at a ratio of 10 : 1, and the drugs were tested in IC 25 concentrations. Drug activity shown in was determined on the basis of both the percentage of infected cells and the number of amastigotes per infected cell in treated and untreated cultures in methanol-fixed and Giemsa-stained preparations.
On day 10, the rate of host cell infection reached its maximum in the control experiment. When the selected compounds were added to the infected cells, the infection rate significantly decreased with respect to the control for all of the compounds tested, leading to inhibition values varying from 44 % to 72 %. In case of L. infantum, the five tested compounds (1 a, 1 f, 3 c, 3 d and 4 b) were more effective than Glucantime, which showed a 29 % inhibition, with 47 %, 47 %, 51 %, 56 % and 44 % inhibition, respectively ( Figure 2).
Intracellular replication of T. cruzi amastigotes was determined in a similar way (at a ratio of 10 : 1, and the drugs in IC 25 concentrations). Only two of the tested compounds (3 b and 3 c) were more effective than Benznidazole, since the two compounds, showing inhibition values of 40 % and 57 %, were found to be more effective than BZN, which only achieved 27 % inhibition on day 10. (Figure 5)

Inhibitory effect on L. braziliensis, L. donovani, L. infantum and T. cruzi Fe-SOD enzymes
Previously we had found that benzo[g]phthalazine and phthalazine derivatives containing sp 2 or sp 3 nitrogen atoms in the side chains and other pyrazole-containing related structures were able to inhibit the activity of the Fe-SOD enzyme of three of these parasites, T. cruzi, L. infantum and L. braziliensis. In all   cases, their inhibiting features were found to be related to the high complexing potentiality of the polyaminic structures assayed, since the environment of the active site could be modified by complexation. [20][21][22][23][24]28] Therefore, the effects of the compounds showing better selectivity index against each of the four parasites under study on the corresponding parasite Fe-SOD, were assayed at concentrations ranging from 1 μM to 100 μM. In each case, epimastigote (Trypanosoma) or promastigote (Leishmania)   forms of the parasite, which excreted Fe-SOD when cultured in a medium lacking inactive fetal calf serum (FCS), were used. [29] The inhibition data obtained are shown in Figures 6-9, where the corresponding IC 50 values are included in order to make the interpretation of results easier. For comparison, Figure 10 shows the effect of the same compounds on CuZn-SOD obtained from human erythrocytes.    According to Figure 6, the most active compounds against L. infantum, 3 d and 4 b, inhibited the enzymatic activity to more than 80 % extent, being especially noteworthy compound 3 d with a IC 50 value of 7,73 μM. Results shown in Figure 7 indicate that the same both compounds. 3 d and 4 b, inhibited the L. braziliensis enzyme activity at approximately 100 % extent, being especially noteworthy in this case compound 4 b with a IC 50 value of 0,09 μM. Inhibition of L. donovani enzyme shown in Figure 8 shows some differences in relation to the preceding data. In this case, only compound 3 c reaches enzyme activity inhibition at 100 % extent with a IC 50 value of 30,54 μM. Finally, data shown in Figure 9 show that, in this case, is 3 b the best compound although it reaches enzyme activity inhibition only at approximately 50 % extent with a IC 50 value of 66,01 μM.
A simple visual comparison of graphs in Figures 6 and 9 and in Figure 10 shows that the parasitic enzyme is inhibited, while the effect caused in CuZn-SOD is much lower in comparison. Compounds 3 b and 3 d caused the highest enzymatic inhibitions, which could explain their noteworthy in vitro results.

Molecular modeling
Fe-SOD enzymes are usually formed by two monomers, both of which contain a non-heme iron active site. This iron atom is coordinated by three histidine units, an aspartate group and an axial H2O or HO-ligand, in trigonal-bipyramidal structure. This structure is supported by an essential H-bonding network, comprised of amino acids and extending across the interface to the other monomer of the Fe-SOD dimer. [27] In previous work with related phthalazine derivatives, [22,[28][29][30][31][32] tentative molecular modelling studies on their mode of interaction with the enzyme were performed. Some correlation between the experimental Fe-SOD inhibitory activity and theoretical perturbation of the geometry and H-bonding patterns at the enzyme core in the parasites was found, suggesting that these highly complexing polyaminic heterocycles could affect Fe-SOD performance by interacting with the H-bonding amino acid system of the ironbased moiety. [24,28] On this basis we have modeled the interaction of compound 3 d with Leishmania Fe-SOD, as it is an especially effective compound in the inhibition of Fe-SOD.
The tertiary structures of superoxide dismutases are very similar, mainly among iron-and manganese-dependent ones. Moreover, the active site residues are conserved, suggesting a   common mechanism of action. [29] Having this into account, the Fe-SOD enzyme structure used for our theoretical studies was obtained from the Brookhaven protein data bank (entry 4F2N corresponding to L. major, as it is the only published structure for a Leishmania species [33] ).The Amber force field implemented in Hyperchem 8. [34] following the methodology specified in the Experimental Section, have been used as in our previous studies. The active site of the enzyme, with the H-bonding system outlined above displayed as dashed lines and the solvent ligand in the foreground linked to the iron atom, is shown in Figure 11A. The key interactions with Gln69 and Asp156 are also clearly seen. Each compound was then buried into one monomer of the Fe-SOD with the imidazole ring pointing towards the metal ion, in a slow motion to prevent the global structure distortion, and the energetically most favored disposition was identified in each case ( Figure 11B for 3 d as an example). As a consequence of the approach of the molecule, the amino acids pattern surrounding the iron atom was distorted, being displaced apart from their initial positions and so the H-bonding pattern appeared remarkably changed. It is noteworthy to point out that a quite similar distortion could be obtained when the pyrimidine ring also present in 3 d pointed towards the metal ion ( Figure 11C).

Synthesis of 1 a-b, 1 e-f, 3 a-d and 4 a.
A solution of the corresponding monochloroazine or monochlorobenzoazine, the corresponding aliphatic amine and trimethylamine in the adequate solvent was heated at 100-120°C for several hours. The reaction mixture was cooled to room temperature and the solvent removed under reduced pressure. The residue was purified by column

Synthesis of 4 b-d.
A solution of the corresponding monochloroazine and the corresponding aliphatic amine in absolute ethanol was heated at 120°C for three hours in a reactor. The reaction mixture was cooled to room temperature and the solvent removed under reduced pressure. The residue was purified by column chromatography with chloroform/methanol 8;2 as eluent to obtain the desired product.

Synthesis of compounds 1 c-d.
A solution of the dichloropyridazine, the corresponding aliphatic amine and trimethylamine in xylene was heated at 110°C for several hours. The reaction mixture was cooled to room temperature and the solvent removed under reduced pressure. The residue was purified by column chromatography with the adequate eluent to obtain the desired product

Synthesis of compounds 2 a-b.
A solution of the dichlorophthalazine, the corresponding aliphatic amine and trimethylamine in absolute ethanol was heated at 100°C for 24 hours in a reactor. The reaction mixture was cooled to room temperature and the solvent removed under reduced pressure. The residue was purified by column chromatography with the adequate eluent to obtain the desired products cultured for 72 h in RPMI and 10 % inactivated foetal bovine serum. The activity of the compounds was determined from the percentage reduction in the number of amastigotes in the treated and untreated cultures in methanol-fixed and Giemsa-stained preparations. The values are the means of four separate determinations. [30] The leishmanicidal or trypanocidal effect was expressed as IC 50 values.

N1,N4-bis[2-(1H-benzo[d]imidazol-2-yl)ethyl]phthalazine-1,4-diamine (2 a) and N-[2-(1H-benzo[d]imidazol-2-yl)ethyl]-4-chloroph-
Infectivity assay: Vero cells were cultured in RPMI medium as described above. Afterwards, the cells were infected in vitro with metacyclic forms of L. infantum, L. braziliensis, L. donovani or T. cruzi at a ratio of 10 : 1. The test compounds (IC 25 concentrations) were added immediately after infection and incubated for 12 h at 37°C in a 5 % CO 2 atmosphere. The extracellular parasites and the test compounds were removed by washing and the infected cultures were grown for 10 days in fresh medium. Fresh culture medium was added every 48 h. The activity of each compound tested was determined from the percentage of infected cells and the number of amastigotes per infected cell in treated and untreated cultures in the methanol-fixed and Giemsa-stained preparations. The percentage of infected cells and the mean number of amastigotes per infected cell were determined by analyzing more than 100 host cells distributed throughout randomly chosen microscopic fields. The values are the means of four separate determinations. The number of trypomastigotes in the medium was determined microscopically (OLYMPUS CX41) for parasite counting using the Neubauer hemocytometric chamber (a dilution of 1 : 100 in PBS was necessary to get into the range of counting). The activity (% of parasites reduction) was compared with the control. [30]

SOD enzymatic inhibition
Leishmania spp. promastigotes and T. cruzi epimastigotes were grown in tissue-culture flasks in axenic medium trypanosomes liquid (MTL) medium (Hank's Balanced Salt Solution -HBSS (Gibco®), supplemented with 10 % heat-inactivated foetal bovine serum (FBS) at 26°C until reaching a population of approximately 1 × 10 7 parasites per mL. Cells were harvested at the logarithmic growth phase by centrifugation (1500 g for 10 min at room temperature). These conditions are stressful for parasites and lead to SODe excretion. These cultures were maintained at 27°C for 24 h.
After 24 h, the promastigote culture was centrifuged (1500 × g for 10 min) and the supernatant was filtered (Minisart®, F 20 mm). The filtered supernatant was precipitated to ice-cold ammonium sulphate at 35 % salt concentration.
Following centrifugation, the resultant supernatant was then treated with 85 % ice-cold ammonium sulphate and the second precipitate collected. The resulting precipitate was finally dissolved in 2.5 mL of distilled water and desalted by chromatography in a Sephadex G-25 column (GE Healthcare Life Sciences®, PD 10 column), previously equilibrated with 25 mL of distilled water, bringing it to a final volume of 3.5 mL (P85e fraction).
Protein concentrations were determined using the Bradford method. [42] Iron and copper-zinc superoxide dismutase (Fe-SOD and CuZn-SOD) activities were determined using the method described by Beyer and Fridovich, [43] which measures the reduction in nitroblue tetrazolium (NBT) by superoxide ions. Into each bucket, 845 μL of Stock solution [3 mL of L-methionine (300 mg, 10 mL À l ), 2 mL of NBT (1.41 mg, 10 mL À 1 ) and 1.5 mL of Triton X-100 1 % (v/v)] were added, along with 30 μL of the parasite homogenate fraction, 10 μL of riboflavine (0.44 mg, 10 mL À l ), and an equivalent volume of the different concentrations of the compounds tested. Five different concentrations were used for each product: 1, 2.5, 5, 12.5 and 25 μM (equivalent to 5 μL, 12.5 μL, 25 μL, 62 μL and 125 μL, respectively, of the Stock solution). In the control experiment the volume was made up to 1000 μL with 50 mM potassium phosphate buffer (pH 7.8, 3 mL), whereas 30 μL of the parasite homogenate fraction was added to the mixtures containing the compounds. Then, the absorbance (A 0 ) was measured at 560 nm in a spectrophotometer. Following this, each bucket was illuminated with UV light for 10 min under constant stirring and the absorbance (A 1 ) was measured. The human CuZn-SOD, coenzymes and substrates used in these assays were obtained from Sigma Chemical Co. The data obtained were analyzed using the Newman-Keuls test.

Molecular modeling
Molecular modeling studies were carried out using the AMBER method implemented in Hyperchem 8.0 package, [35] modified by the inclusion of appropriate parameters. [44] Starting structure for compound 3 d were built by using Hyperchem capabilities. Its geometry was minimized to a maximum energy gradient of 0.1 kcal/mol with the AMBER force field, using the Polak-Ribiere (conjugate gradient) minimizer, and'simulated annealing' procedure was used to cover all conformational space. The most stable extended geometry was used in the calculations of interaction with the enzyme. To mimic the conditions used in the activity measurements, i. e., water as solvent, all calculations were carried out in vacuo with distance dependent dielectric constant value. Charge assignations for all atoms was done by means of ab initio calculations using STO-3G basis set, as it is compatible with AMBER force field, prior to energy minimization using AMBER. The Fe-SOD enzyme structure was obtained from the Brookhaven protein data bank (4F2N for Leishmania) and its energy minimized in the same way. Interaction studies were performed starting from structures with the compound positioned in the border of the enzyme cavity. Entering the cavity was forced using a restraint to the NÀ Fe distance, slowly decreasing this distance, and letting the complex achieve the minimum energy conformation with no restraints, for all the small driving steps, using the same conditions mentioned above.