Synthesis, In Silico, and In Vitro Evaluation of Anti-Leishmanial Activity of Oxadiazoles and Indolizine Containing Compounds Flagged against Anti-Targets

Due to the lack of approved vaccines against human leishmaniasis and the limitations of the current chemotherapy inducing side effects and drug resistance, development of new, effective chemotherapeutic agents is essential. This study describes the synthesis of a series of novel oxadiazoles and indolizine-containing compounds. The compounds were screened in silico using an EIIP/AQVN filter followed by ligand-based virtual screening and molecular docking to parasite arginase. Top hits were further screened versus human arginase and finally against an anti-target battery to tag their possible interactions with proteins essential for the metabolism and clearance of many substances. Eight candidate compounds were selected for further experimental testing. The results show measurable in vitro anti-leishmanial activity for three compounds. One compound with an IC50 value of 2.18 µM on Leishmania donovani intramacrophage amastigotes is clearly better positioned than the others as an interesting molecular template for further development of new anti-leishmanial agents.

Synthesis of the 3-[3,4-(methylenedioxy)phenyl)]-5-(methoxymethyl)-1,2,4-oxadiazole 8 was achieved in two steps and 90% total yield by the reaction of 3,4-(methylenedioxy)benzonitrile with hydroxylamine to afford quantitatively amidoxime 6 followed by the reaction with methoxyacetyl chloride under microwave irradiation (Scheme 2). This compound was prepared during a small molecule library synthesis program, and the protocols established to allow the automation and the parallelization of reactions. The first step of the procedure was applied to 3-nitrobenzonitrile, affording also in quantitative yield the corresponding 3-nitrobenzamidoxime. Scheme 1. Automated synthesis of acylhydrazones 1-5; for compounds that were isolated: R1 = furan, thiophene, pyridine; R2 = 4-methyl-thiazole, 2-bromo-thiophene, benzothiazole. 1,2,4-Oxadiazoles can be considered as one of the most important 5-membered heteroaromatic rings found in many pharmaceutical compounds. Among the various synthetic approaches reported in the literature [24], one concerns reaction under conventional or non-conventional methods of amidoximes with suitably activated acid derivatives [25].
Synthesis of the 3-[3,4-(methylenedioxy)phenyl)]-5-(methoxymethyl)-1,2,4-oxadiazole 8 was achieved in two steps and 90% total yield by the reaction of 3,4-(methylenedioxy)benzonitrile with hydroxylamine to afford quantitatively amidoxime 6 followed by the reaction with methoxyacetyl chloride under microwave irradiation (Scheme 2). This compound was prepared during a small molecule library synthesis program, and the protocols established to allow the automation and the parallelization of reactions. The first step of the procedure was applied to 3-nitrobenzonitrile, affording also in quantitative yield the corresponding 3-nitrobenzamidoxime. Scheme 1. Automated synthesis of acylhydrazones 1-5; for compounds that were isolated: R1 = furan, thiophene, pyridine; R2 = 4-methyl-thiazole, 2-bromo-thiophene, benzothiazole. 1,2,4-Oxadiazoles can be considered as one of the most important 5-membered heteroaromatic rings found in many pharmaceutical compounds. Among the various synthetic approaches reported in the literature [24], one concerns reaction under conventional or non-conventional methods of amidoximes with suitably activated acid derivatives [25].
Synthesis of the 3-[3,4-(methylenedioxy)phenyl)]-5-(methoxymethyl)-1,2,4-oxadiazole 8 was achieved in two steps and 90% total yield by the reaction of 3,4-(methylenedioxy)benzonitrile with hydroxylamine to afford quantitatively amidoxime 6 followed by the reaction with methoxyacetyl chloride under microwave irradiation (Scheme 2). This compound was prepared during a small molecule library synthesis program, and the protocols established to allow the automation and the parallelization of reactions. The first step of the procedure was applied to 3-nitrobenzonitrile, affording also in quantitative yield the corresponding 3-nitrobenzamidoxime.  Scheme 1. Automated synthesis of acylhydrazones 1-5; for compounds that were isolated: R1 = furan, thiophene, pyridine; R2 = 4-methyl-thiazole, 2-bromo-thiophene, benzothiazole. 1,2,4-Oxadiazoles can be considered as one of the most important 5-membered heteroaromatic rings found in many pharmaceutical compounds. Among the various synthetic approaches reported in the literature [24], one concerns reaction under conventional or non-conventional methods of amidoximes with suitably activated acid derivatives [25].
Synthesis of the 3-[3,4-(methylenedioxy)phenyl)]-5-(methoxymethyl)-1,2,4-oxadiazole 8 was achieved in two steps and 90% total yield by the reaction of 3,4-(methylenedioxy)benzonitrile with hydroxylamine to afford quantitatively amidoxime 6 followed by the reaction with methoxyacetyl chloride under microwave irradiation (Scheme 2). This compound was prepared during a small molecule library synthesis program, and the protocols established to allow the automation and the parallelization of reactions. The first step of the procedure was applied to 3-nitrobenzonitrile, affording also in quantitative yield the corresponding 3-nitrobenzamidoxime.  1,2,4-Oxadiazoles can be considered as one of the most important 5-membered heteroaromatic rings found in many pharmaceutical compounds. Among the various synthetic approaches reported in the literature [24], one concerns reaction under conventional or non-conventional methods of amidoximes with suitably activated acid derivatives [25].
Synthesis of the 3-[3,4-(methylenedioxy)phenyl)]-5-(methoxymethyl)-1,2,4-oxadiazole 8 was achieved in two steps and 90% total yield by the reaction of 3,4-(methylenedioxy)benzonitrile with hydroxylamine to afford quantitatively amidoxime 6 followed by the reaction with methoxyacetyl chloride under microwave irradiation (Scheme 2). This compound was prepared during a small molecule library synthesis program, and the protocols established to allow the automation and the parallelization of reactions. The first step of the procedure was applied to 3-nitrobenzonitrile, affording also in quantitative yield the corresponding 3-nitrobenzamidoxime. Scheme 1. Automated synthesis of acylhydrazones 1-5; for compounds that were isolated: R1 = furan, thiophene, pyridine; R2 = 4-methyl-thiazole, 2-bromo-thiophene, benzothiazole. 1,2,4-Oxadiazoles can be considered as one of the most important 5-membered heteroaromatic rings found in many pharmaceutical compounds. Among the various synthetic approaches reported in the literature [24], one concerns reaction under conventional or non-conventional methods of amidoximes with suitably activated acid derivatives [25].

EIIP Filtering
We used the previously developed average quasi valence number/electron ion interaction potential (EIIP/AQVN) criteria for selection of Leishmania arginase compounds [20].

3D QSAR Filtering
39 molecules were obtained after filtering with EIIP criteria and were subjected to prediction of their activity using the above described arginase 3D-quantitative structure-activity relationship (3D-QSAR) model. The two criteria for selection were: (1) partial least square (PLS) scores in the  (2) best ranking by predicted IC 50 values. This filtering gave 10 candidates that were used for docking into the Leishmania arginase structure model, human arginase crystal structure, and off-target (anti-target) affinity calculation.
We used the previously developed average quasi valence number/electron ion interaction potential (EIIP/AQVN) criteria for selection of Leishmania arginase compounds [20].

3D QSAR Filtering
39 molecules were obtained after filtering with EIIP criteria and were subjected to prediction of their activity using the above described arginase 3D-quantitative structure-activity relationship (3D-QSAR) model. The two criteria for selection were: 1) partial least square (PLS) scores in the vicinity of compounds from the model (Figure 1); and 2) best ranking by predicted IC50 values. This filtering gave 10 candidates that were used for docking into the Leishmania arginase structure model, human arginase crystal structure, and off-target (anti-target) affinity calculation.

Arginase Docking
Ten compounds were docked into both the parasite arginase model structure and the crystal structure of human arginase. Docking scores of the best-docked conformations, along with experimental measurements are presented in Table 2. Although PLS prediction and docking score results were promising, the experiments showed significant activity of six compounds. However, due to their toxicity, only one candidate, compound 2, is acceptable thanks to its selectivity index (SI, CC50 for macrophage/IC50 for promastigotes) ≥ 2, with an IC50 value on intramacrophage amastigotes < 5 µM. The highest ranked docking conformation of this compound in the Leishmania arginase model is presented in Figure 2.

Arginase Docking
Ten compounds were docked into both the parasite arginase model structure and the crystal structure of human arginase. Docking scores of the best-docked conformations, along with experimental measurements are presented in Table 2. Although PLS prediction and docking score results were promising, the experiments showed significant activity of six compounds. However, due to their toxicity, only one candidate, compound 2, is acceptable thanks to its selectivity index (SI, CC 50 for macrophage/IC 50 for promastigotes) ≥ 2, with an IC 50 value on intramacrophage amastigotes < 5 µM. The highest ranked docking conformation of this compound in the Leishmania arginase model is presented in Figure 2.

Anti-Target Interaction Matrix
The top eight compounds from the filtering were then assessed against the anti-target battery. The results of the docking of all the final compounds against the battery of five anti-targets are shown in Figure 3 (full table of docking scores Table 3).
PXR SULT CYP CYP CYP Total

Anti-Target Interaction Matrix
The top eight compounds from the filtering were then assessed against the anti-target battery. The results of the docking of all the final compounds against the battery of five anti-targets are shown in Figure 3 (full table of docking scores Table 3).

Anti-Target Interaction Matrix
The top eight compounds from the filtering were then assessed against the anti-target battery. The results of the docking of all the final compounds against the battery of five anti-targets are shown in Figure 3 (full table of docking scores Table 3). There was broad general agreement between the five anti-targets. None of the compound's docking score surpassed that of the threshold for CYP P450 2a6, 2c9, or 3a4, which may be an indication of the relative size of the ligands. There were more interactions found with the anti-targets PXR SULT CYP 2a6 CYP 2c9 CYP 3a4  There was broad general agreement between the five anti-targets. None of the compound's docking score surpassed that of the threshold for CYP P450 2a6, 2c9, or 3a4, which may be an indication of the relative size of the ligands. There were more interactions found with the anti-targets PXR and SULT. Compounds 3 and 5 had the highest combined score of 1.0 for all anti-targets combined, while most compounds had even lower interactions.
All of the compounds had zero PAINS flags, passed Lipinksi's rule-of-five, and were predicted to be soluble or moderately soluble, have high gastrointestinal absorption, and potential (using a support-vector-machine) CYP 2a6 binding for 2, 3, 4, 5, and CYP 3a4 for 8, according to filters [28].

In vitro Evaluation of Anti-Leishmanial Activity
Finally, we selected eight hit compounds for experimental testing. Since isolated arginase was not available, the selected compounds were assayed for their in vitro inhibition activity against axenic amastigote and intramacrophage amastigote forms of Leishmania donovani (Table 3). A broad range of activities against axenic and intramacrophage amastigotes forms of Leishmania donovani was found with IC 50 values in the range between 1-2 and 55-61 µM. The compounds exhibited similar activities on both axenic amastigotes and intramacrophage amastigotes. The most potent inhibitors were also slightly toxic with CC 50 values in the range from 4 to 16 µM.
Regarding structure-activity relationships, the replacement of a thiophene group (compound 4) by a pyridine group (compound 5) was responsible for a loss of activity by a factor of five and also a strong reduction of cytotoxicity. Among the tested compounds, one compound, 2, is the most active despite having a not negligible toxicity. Its chemical structure has two thiophene groups. The replacement of a thiophene group (compound 2) by a furan group (compound 3) led to a total loss of activity and cytotoxicity. The replacement of the bromothiophene group (compound 2) by a benzothiazole group (compound 4) was responsible for a five-fold reduction of the anti-leishmanial activity and four-fold reduction of cytotoxicity. The replacement of bromothiophene (compound 3) by a methylbenzothiazole group (compound 1) enhanced both the antileishmanial activity and cytotoxicity. In the phenotypic based screening, the most promising compound 2 (Table 3) has an IC 50 value of 2.18 µM, 20 times higher than that of amphotericin B, the reference compound. Regarding the selectivity index (SI) values, compound 2 has an SI of 2, whereas amphotericin B had an SI of 48 against Leishmania axenic amastigotes. It is also less than four times higher than the reference drug miltefosine (0.31 µM) [29]. The obtained in vitro results clearly require additional pharmacomodulations to reduce the cytotoxicity and enhance the antileishmanial activity in order to achieve better selectivity index values.

Discussion
Inhibition of enzymes of the polyamine-trypanothione metabolism including arginase is considered as one of the best options for the treatment of Leishmania since many of these enzymes passed both target validation and chemical validation [10]. In the quest for potential treatment options against leishmaniasis, increasing interest has been shown for N-acylhydrazone, oxadiazole, and indolizine containing compounds. These types of compounds targeting arginase represent an interesting strategy in the search for a new anti-leishmanial treatment.
In our hands, N-acylhydrazones were prepared on an automated platform where reaction conditions and purifications by filtration of the solid compounds were parallelized for 50 compounds in each run (about 200 compounds synthetized in four batches). In that respect, reaction conditions were generalized, and for some of the compounds, yields are not optimum. The same is also true for the 1,2,4-oxadiazoles where the reaction conditions were chosen in order to prepare a small-molecule library (150 compounds synthetized). The reaction conditions first afforded the amidoxime compounds quantitatively, then the reaction with activated acid furnished in good yield the 1,2,4 oxadiazoles, where one example is used in the current study. Concerning the indolizine compounds, the azomethine ylide prepared in situ by 1,3 dipolar cycloaddition was chosen. The yields obtained for compounds 11-13 are poor, varying between 25-28% after careful purification. The insolubility of the chloride salts could be the reason for the low yields. As one of the research programs of the group concerns 1,3 dipolar cycloadditions using this type of ylides, efforts are currently oriented to the synthesis of indolizine-bearing derivatives under non-conventional methods in order to obtain focused small libraries of these compounds.
The compounds thus obtained for this study were subsequently screened. The ten selected compounds from our study with favorable in silico interaction profiles with arginase and against the anti-targets were favorable for further experimental testing and represent better initial points than screening compounds at random. Three compounds were found to be active against Leishmania donovani. One product, compound 2, is better than the others as an interesting molecular template for further development of new anti-leishmanial agents due to its observed experimental activity against parasite amastigotes, better selectivity index, and predicted interactions with targets and anti-targets. Interestingly, the compound that had the best selectivity index, the most promising compound 2, had a docking score that was approximately 0.8 kcal/mol stronger for the parasite arginase than for human arginase. This may also indicate that a larger degree of selectivity for a compound may be picked up by the procedure carried out here.
Considering the mechanism of action of both chemical series, the results obtained in this paper prompt us to develop further pharmacomodulations in order to diminish the cytotoxicity and enhance the anti-leishmanial activity. In any case, despite the level of cytotoxicity of compound 2 being in a similar range to that of the reference compound AmB, the obtained results justify the determination of the maximal tolerated dose in mice, and then, the in vivo evaluation of compound 2 on the L. donovani/BALB/c mice model.

Chemistry and Physico-Chemical Analyses
Melting points (m.p.) were determined using a Mettler Toledo MP50 system and were uncorrected. 1 H and 13 C NMR spectra were recorded in CDCl 3 and/or DMSO-d6 using a Bruker AC 300 ( 1 H) or 75 MHz ( 13 C) instruments, except for compounds 1, 2, 4, 6, and 7: 13 C-NMR analyses were conducted with a Bruker AVANCE500 at 298 K (125.75 MHz). Chemical shifts are given in δ parts per million (ppm) and referenced to external TMS.
Automated syntheses were carried out on an Accelerator SLT-106 workstation from Chemspeed and microwave assisted reactions on a SWave workstation from Chemspeed equipped with a Biotage Initiator reactor.

Ligands
The source for data was internal libraries of synthetized oxadiazoles and indolizine-containing compounds.

Virtual Screening
Starting with a filter based on EIIP/AQVN values and subsequently, 3D QSAR filtering, and arginase docking, hit compounds were docked into anti-targets involved in the metabolism of compounds in order to tag their possible interactions.

EIIP/AQVN
AQVN and the EIIP can give indication on long-range biomolecule interaction (over 0.5 nm) [30], based on the general model pseudopotential [31] EIIP = 0.25 Z* sin(1.04 π Z*)/2π, where Z* is the average quasi-valence number (AQVN): where Zi is the valence number of the ith atomic component, ni is the number of atoms of the ith component, m is the number of atomic components in the molecule, and N is the total number of atoms. EIIP values are calculated according to equations 1 and 2 are expressed in Rydberg units (Ry). Similarity in AQVN and EIIP values may give a clue on a common therapeutic target, thus setting up criteria for virtual screening of molecular libraries for compounds with similar therapeutic properties [20].

Ligands
The source for data was internal libraries of synthetized oxadiazoles and indolizine-containing compounds.

Virtual Screening
Starting with a filter based on EIIP/AQVN values and subsequently, 3D QSAR filtering, and arginase docking, hit compounds were docked into anti-targets involved in the metabolism of compounds in order to tag their possible interactions.

EIIP/AQVN
AQVN and the EIIP can give indication on long-range biomolecule interaction (over 0.5 nm) [30], based on the general model pseudopotential [31] EIIP = 0.25 Z* sin(1.04 π Z*)/2π, where Z* is the average quasi-valence number (AQVN): Z* = ∑ m(ni Zi / N), where Zi is the valence number of the ith atomic component, ni is the number of atoms of the ith component, m is the number of atomic components in the molecule, and N is the total number of atoms. EIIP values are calculated according to equations 1 and 2 are expressed in Rydberg units (Ry). Similarity in AQVN and EIIP values may give a clue on a common therapeutic target, thus setting up criteria for virtual screening of molecular libraries for compounds with similar therapeutic properties [20].

Arginase
The crystal structure of Leishmania mexicana with ABH (4iu0.pdb) was used to generate a model for L. amazoniensis. Docking was performed with Autodock 4.6.2. The Lamarckian GA method was used to perform the conformational search.

Anti-Targets
The docking for the anti-targets was carried out with Glide XP [32] and proteins with resolution ≤ 2.6 Å (1m13, 2a3r, 1z10, 1og5, 1tqn) [33,34]. The thresholds for determining a strong interaction were set to −7.7, −6.3, −7.6, −8.7, and −7.5 kcal/mol. An estimate of the binding was changed to interaction codes: where ∆Gref is the docking score of the protein with co-crystallized active reference ligand, and ∆G is the docking score for a ligand bound to that protein binding site. Shades were then set to white, grey, and black.

Macrophage
RAW 264.7 macrophages were grown with 5% CO 2 in DMEM complete medium containing Dulbecco's Modified Eagle's Medium (DMEM) with 100 U/mL penicillin-streptomycin, and 10% HIFBS at 37 • C. 4.6.3. In Vitro Anti-Leishmanial Compound Testing on Axenic and Intramacrophage Amastigotes: Previously described protocols [35] were adapted for evaluation of compounds on L. donovani. For axenic amastigotes, two-fold serial dilutions of the compounds were done in 96-well microplates with 100 µL of complete medium (see above). Axenic amastigotes were then added to each well at a density of 106 mL in a 200 µL final volume. After 72 h of incubation with 5% CO 2 at 37 • C, 20 µL of resazurin (450 µM) was added to each well and further incubated with 5% CO 2 at 37 • C for 24 h in the dark. Resazurin is reduced to resorufin in living cells and can be then monitored by measuring