Synthesis of carboxyimidamide-substituted benzo ij c ] ij 1 , 2 , 5 ] oxadiazoles and their analogs , and evaluation of biological activity against Leishmania donovani †

Med. Chem. Commu This journal is © The Royal Society of Chemistry 2015 a Faculty of Pharmacy, Division of Pharmaceutical Chemistry and Technology, University of Helsinki, Viikinkaari 5 E, P. O. Box 56, FI-00014 Helsinki, Finland. E-mail: paula.kiuru@helsinki.fi b Department of Microbiology and Molecular Genetics, IMRIC, Hebrew UniversityHadassah Medical School, P. O. Box 12272, Jerusalem 9112102, Israel † Electronic supplementary information (ESI) available: Synthesis procedures for compounds 6–13, 15–18, 21–39. See DOI: 10.1039/c5md00119f Cite this: Med. Chem. Commun., 2015, 6, 1673


Introduction
Leishmaniasis is a spectrum of human diseases caused by at least 20 different species of protozoan parasites.These parasites are responsible for three major diseases: cutaneous, visceral, and mucocutaneous.Over 300 million people are at risk. 1 Visceral leishmaniasis (VL), caused primarily by L. donovani (anthroponotic) or L. infantum/L.chagasi (zoonotic), is fatal if left untreated.There are an estimated 300 000 cases of VL per year, and 20 000 to 40 000 deaths.Over 90% of new cases occur in six countries: Bangladesh, Brazil, Ethiopia, India, South Sudan, and Sudan.Although two new drugs, miltefosine and paromomycin, are currently in use and combination therapy is becoming more common, 2 an urgent need for new antileishmanial chemotypes persists.The major issues to be confronted in Leishmania treatment are the toxicity of primary medicines and the development of parasite resistance.
In addition, poor efficacy in the case of visceral leishmaniasis/HIV coinfection has raised the need for new antileishmanial treatments. 3n our previous study, 2-arylbenzimidazole and benzoĳc]-ĳ1,2,5]oxadiazole derivatives showed activity against the intracellular Gram-negative bacterium Chlamydia pneumoniae. 4 In that study, a similarity-based model of C. pneumoniae dimethyladenosine transferase was used to virtually screen compounds from commercially available databases against C. pneumoniae.The 2-arylbenzimidazoles found were then tested and proven active against L. donovani.5 This prompted us to consider benzoĳc]ĳ1,2,5]oxadiazole-derived compounds as a new family of antileishmanial agents.In addition, benzoxadiazoles and their N-oxides have been previously studied as antibiotic and antiparasitic agents.6 In this study, a facile synthesis route to carboxyimidamide-substituted benzoxadiazoles was developed, a set of 25 benzoĳc]ĳ1,2,5]oxadiazole derivatives and other structurally related compounds was synthesized and evaluated as antileishmanial agents.

Chemistry
Initially, benzoĳc]ĳ1,2,5]oxadiazole-5-carbonitrile 4 was to be synthesized from 5-chloro-or 5-bromobenzoĳc]ĳ1,2,5]oxadiazole in a microwave-assisted reaction using NaCN, CuCN, or K 4 ĳFeĲCN) 6 ], 7 but these syntheses failed to produce the target nitrile.Instead the synthesis of benzoĳc]ĳ1,2,5]oxadiazole derivatives was started from the commercially available 4-amino-3-nitrobenzoic acid (Scheme 1).It was converted to benzoĳc]ĳ1,2,5]oxadiazole-5-carboxylic acid (1) by using a two-step procedure via the N-oxide intermediate that was produced in the presence of sodium hypochlorite in an alkaline EtOH-H 2 O solution and subsequently reduced to the carboxylic acid 1. 8 Next, compound 1 was converted to the primary amide 3 via the acyl chloride 2 in the presence of aqueous ammonia in 1,4-dioxane.The reaction of the obtained primary amide 3 with trifluoroacetic anhydride and Et 3 N in THF 9 gave the corresponding nitrile 4, which was converted to the amidoxime 5 by using hydroxylamine This journal is © The Royal Society of Chemistry 2015 hydrochloride 10 in the presence of Et 3 N in EtOH.The final step to obtain the derivatives (6-18, 24) was carried out in the presence of the substituted phenyl isocyanates in THF or CHCl 3 .The products 21-23 were synthesized in a similar manner by using a solution of Et 3 N in THF.Compound 20 was synthesized from the acyl chloride 2 via the hydroxamic acid 19.The final products 33-39 were synthesized from the commercially available nitriles by using the methods described above (Scheme 2).Finally, the related benzimidazole derivative 25 was formed from 3,4-diaminobenzonitrile in refluxing formic acid. 11

Evaluation of antileishmanial activity
The activity of the new compounds was determined by using a fluorescent viability microplate assay with L. donovani axenic amastigotes and alamarBlue. 12All compounds were initially screened at 50 μM, and the most active derivatives sequentially tested at lower concentrations (15 and 5 μM).
Replacing HN of the heteroatom containing chain with oxygen or carbon atom (compounds 22 and 23) significantly reduced antileishmanial activity to 27 and 22%, respectively, at 50 μM.Similarly, removal of the aniline NH (compound 21) reduced activity, but this compound still showed more activity (51% growth inhibition) than carboxy 22 or benzylic 23 derivatives.Moreover, the cyclohexane derivative 24 displays leishmanial inhibition in the absence of an aniline moiety.Replacement of the amino group in the imidamide 20 with a carbonyl group increased inhibition to 96% and 68% at concentrations of 50 μM and 15 μM, respectively.
Finally, a second set of related heterocyclic compounds was synthesized with replacements of the benzoĳc]ĳ1,2,5]oxadiazole moiety.These heterocycles (benzothiophene 36, benzofuran 37, indole 38, and 1,3-benzodioxole 39) showed an increase in antileishmanial activity at 50 μM compared to the benzoxazole derivative 6.This suggests that the benzoĳc]-ĳ1,2,5]oxadiazole ring is not crucial to antileishmanial activity.In addition, the phenyl and naphthalene derivatives 33 and 34 significantly inhibited growth of L. donovani amastigotes (78% and 5% for 33, and 99% and 25% for 34, at concentrations of 50 μM and 15 μM, respectively).Good inhibition of compound 34 can be hypothesized to result from increased lipophilicity of the compound.Moreover, the phenyl derivative 33 is a very interesting compound, because the shorter synthetic route is likely to increase the prospects for further preparation of new antileishmanial derivatives of this chemotype.
Further evaluation of the most promising derivatives 14 and 20 revealed low 50 values on axenic amastigotes, 4.2 and 8.1 μM respectively; moderate cytotoxicity on the human THP-1 macrophage cell line, 79.9 and 33.7 μM respectively; and no cytotoxicity against murine fibroblasts at the highest concentration tested, 300 μM (Table 2).Values for amphotericin B, a reference compound used to treat leishmaniasis, are given for comparison.When tested at 5 μM on infected macrophages, a concentration non-toxic for the macrophage cell line, both compounds, 14 and 20, were still active, 33.1 and 19.9% amastigote growth inhibition respectively, albeit lower than that observed with axenic amastigotes.The EC 50 for 14, the most active and least cytotoxic derivative, on intracellular amastigotes in infected macrophages was 5.92 μM similar to that observed using axenic amastigotes.

Conclusion
The developed synthesis route to carboxyimidamidesubstituted benzoĳc]ĳ1,2,5]oxadiazoles and related derivatives facilitates access to a compound library and determination of the structure-activity relationships for this antileishmanial heterocyclic chemotype.
Carboxyimidamide-substituted benzoxadiazole derivative 14 is the most promising compound of this study, demonstrating good antileishmanial inhibition activity in infected macrophages and, remarkably, no signs of cytotoxicity.Although mechanism of action of the compounds is not known yet, it is worth continuing the development of these compounds to explore further prospects of these compounds.

Biology
Antileishmanial evaluation using axenic amastigotes.L. donovani (MHOM/SD/1962/1S-Cl2d) was used in all bioassays.Screening of the compounds for antileishmanial activity using axenic amastigotes was carried out using the alamarBlue (AbD Serotec, Oxford, UK) viability assay, as previously described, similar to that reported for leishmanial promastigotes. 12Axenic amastigotes were grown at 37 °C in a 5% CO 2 incubator in complete RPMI 1640 containing 20% fetal calf serum, pH 5.5. 13Compounds to be assayed were diluted in the complete amastigote medium containing 1% DMSO to twice the final concentration used in the assays, and were aliquoted in triplicate (125 μL per well) into 96-well flat-bottom plates (Nunc, Roskilde, Denmark).For determination of the EC 50 , final concentrations in triplicate spanning 300 to 0.14 μM were used.Amastigotes (5.0 × 10 5 cells per mL; 125 μL per well) were added to each well and incubated for 24 h at 37 °C in a 5% CO 2 incubator.The alamarBlue viability indicator was added (25 μL per well) and the plates incubated for an additional 24 h at which time the fluorescence (λ ex = 544 nm; λ em = 590 nm) was measured in a microplate reader (Fluoroskan Ascent FL, Finland).Complete medium, both with and without DMSO, was used as negative controls (0% inhibition of amastigote growth).Amphotericin B (Sigma-Aldrich, St. Louis MO), a drug used to treat VL, was included as a positive control on each plate and gave >90% inhibition of parasite growth at 1.5 μM.
Cytotoxicity.EC 50 was determined on NIH/3T3 fibroblasts or THP-1 monocyte cells using the alamarBlue viability indicator as follows.Cells in DMEM medium plus 10% fetal calf serum and antibiotics were aliquoted in triplicate (fibroblast or THP-1 at 8 × 10 5 or 6.4 × 10 4 cells per well respectively; 125 μL per well) into 96-well plates (Nunc, Roskilde, Denmark).Compounds to be assayed were diluted in the same medium containing 1% DMSO at twice the final concentration used in the assays (300 to 0.14 μM for fibroblasts or 150 to 0.10 μM for THP-1), added to the cells (125 μL per well), and the plates incubated for 48 h at 37 °C in a 5% CO 2 incubator.The alamarBlue viability indicator was added (25 μL per well) and the plates incubated for an additional 3-5 h at which time the fluorescence (λ ex = 544 nm; λ em = 590 nm) was measured.Complete medium, both with and without DMSO, was used as negative controls (0% inhibition).
Activity on intracellular amastigotes.Inhibition of amastigote growth in infected THP-1 cells was measured as previously described. 5In brief, THP-1 cells in complete RPMI-1640 containing 10% fetal calf serum and antibiotics were treated for 24 h with 25 ng mL −1 phorbol 12-myristate-13-acetate (PMA, Sigma-Aldrich, St. Louis, MO).PMA and non-adherent cells were removed with excess warm medium, and stationary-phase Ld:pSSU-int/LUC promastigotes expressing luciferase (5 : 1 parasite/macrophage ratio) used to infect the macrophages.Twenty-four hours later the adherent cells were washed 4-5 times with warm RPMI-1640 alone, and infected macrophages detached by treating with Trypsin EDTA.Infected macrophages (5 × 10 4 cells/100 μL per well) were dispensed in triplicate into white 96-well flat bottom plates (NUNC, Denmark) and the compounds diluted in complete RPMI-1640 containing 1% DMSO final concentration and added in triplicate (50 μL per well).Cultures were incubated for a further 48 h (37 °C, 5% CO 2 ), lysed by the addition of Bright-Glo Luciferase Assay substrate (100 μL per well, Promega, MT, U.S.A.), and chemiluminescence measured This journal is © The Royal Society of Chemistry 2015 using a microplate reader (Fluoroskan Ascent, Thermo Scientific).Amphotericin B (1 μM, >90% inhibition of parasite growth) was included as a positive control.Complete medium, both with and without DMSO, was used as negative controls.Experiments were repeated three times.Calculation of the EC 50 's and statistical analysis were carried out using GraphPad Prism Version 6.0b (GraphPad Software, Inc.San Diego, CA).