New Benzoxazole Derivatives as Antiprotozoal Agents: In Silico Studies, Synthesis, and Biological Evaluation

Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakakah, Aljouf 72341, Saudi Arabia Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt Research Institute of Medicinal and Aromatic Plants, Beni Suef University, Beni Suef 62514, Egypt National Centre for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Oxford, Mississippi 38677, USA Division of Pharmacognosy, Department of Biomolecular Sciences, School of Pharmacy, University of Mississippi, MS 38677, USA Department of Chemistry, College of Science, Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia Biology Department, College of Science, Jouf University, Sakakah, Aljouf 72341, Saudi Arabia Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt


Introduction
Malaria is a lethal illness initiated through the spreading of parasites via the nibbles of infected female mosquitoes. In 2015, the WHO reported that approximately half of the world's population (3.2 billion) was at threat of malaria with approximate deaths of 0.8-1.2 million per year [1]. e African continent bears the brunt of this parasitic disease, with an estimated percentage of 90% of all malarial deaths being children under five years of age [2]. ere is no licensed malaria vaccine available. Due to parasite resistance, the current effective drugs, such as artemisinin combination therapies (ACTs), are becoming less effective and sometimes lead to treatment failure [3] and relapse of malaria. Another life-threatening neglected tropical disease, leishmaniasis, is triggered by several Leishmania parasite genus [4]. e two major types of leishmaniasis in humans are visceral, cutaneous leishmaniasis (CL), and leishmaniasis (VL) [5]. e later tropical disease is primarily triggered by L. donovani and can cause death in all situations if left unprocessed [6]. Alternatively, the growing incidence of infections with multidrug-resistant microbials has become essential problematic healthcare. Specifically, the development of multidrug-resistant pathogenic bacteria such as Staphylococcus epidermidis, vancomycin-resistant Enterococcus, and methicillin-resistant Staphylococcus epidermis is a significant challenge [7][8][9]. e oxazoles derivatives are considered the "isosteres of natural nucleotides" and became the target of many researchers to develop various synthetic analogs with substantial chemotherapeutic activities [10][11][12][13][14]. Many naturally and synthetically bioactive compounds containing benzoxazole scaffold registered to display a wide range of biological actions, for example, antimalarial [6], antileishmanial [6], antiviral [15,16], and antimicrobial [17], along with inhibiting the activity of eukaryotic topoisomerase II enzyme [18][19][20] and activity against multidrug-resistance cancer cells [21][22][23][24][25][26].
A carboxylic polyether antibiotic calcimycin (Figure 1(a)) derived from Streptomyces chartreuses (NRRL 3882) is a natural benzoxazole derivative with very potent activity versus Gram-positive bacteria involving some Micrococcus and Bacillus [27,28]. Also, N-(2-benzoxazole-2ylphenyl)benzamide derivative (Figure 1(b)) is another natural antibiotic isolated from Streptomyces sp. (NRRL 12068) culture with significant antibacterial activity, remarkable activity against Plasmodium falciparum with a percent inhibition of 97 % (conc. 4.8 μg·mL −1 ), and promising antileishmanial activity (three-fold more potent than miltefosine which is an alkylphosphocholine agent with worthy activity against Leishmania) [5]. ese facts have inspired the medicinal chemists to consider the benzoxazole nucleus as a privileged core structure for designing novel chemical entities with potential anti-infective, antiprotozoal, and antimicrobial activities.
In consideration of the aforementioned background and with the extension of our former investigations on benzoxazole moiety and its innate pharmacological activities [14,[29][30][31][32][33], we describe the preparation of a new series of compounds with 3-benzoxazolyl aniline scaffold ( Figure 2). All the analogs were screened for their antimicrobial, antimalarial, antileishmanial, and antitrypanosomal activities.

e General Method for Preparation of Compounds 4a-d.
Triazine (3) (3.58 g, 0.01 mol) was mixed with K 2 CO 3 (2.76 g) and appropriate aniline (0.023 mol) in anhydrous dioxane (25 mL). After heating under reflux for 12 h, the mixture was emptied into ice water, and the separated precipitate was filtrated and crystallized from EtOH to obtain triazine derivatives 4a-d.

e General Method of Chloroketones 5a&b Synthesis.
To a stirred ice-cold solution of 1 (2.10 g, 0.01 mol) in anhydrous dimethylformamide (10 mL), the appropriate chloroacyl chloride (1.12 g) was added slowly, and then, adding K 2 CO 3 (1.38 g) was added. e mixture was further stirred for 24 h at room temperature. At the end of the reaction, the mixture was emptied in ice water; the precipitate was filtrated, left to dry, and crystallized from CH 3 OH to produce chloroketones 5a&b.

In Vitro Antimicrobial Activity. All fungal and bacterial strains have been obtained from the ATCC (American Type Culture Collection).
A total of five fungal strains were selected for testing their vulnerability to the target final compounds. ese strains comprised C. krusei (ATCC 6258), C. albicans (ATCC 90028),C. glabrata (ATCC 90030), Cryptococcus neoformans (ATCC 90113), and Aspergillus fumigatus (ATCC 204305). We also assessed the sensitivity of five bacterial strains to the final target compounds. ese strains comprised methicillin-resistant S. aureus (ATCC 33591) (MRSA), P. aeruginosa (ATCC 27853), E. coli (ATCC 35218), M. intracellulare (ATCC 23068), and S. aureus (ATCC 29213), and vulnerability testing was carried out using the Clinical and Laboratory Standards Institute-(CLSI-) adapted procedure [34]. A modified Franzblau method was applied to test the target compounds reactivity against Mycobacterium intracellulare [35]. All tested compounds were diluted serially utilizing NaCl (0.09%, solution) containing DMSO (20%, dimethyl sulfoxide) and transmitted in duplicate to microplates (96-well). Optical density was applied to correct the microbial inoculum at 630 nm of microbe suspension. In the evaluation protocols, two positive controls were used. Amphotericin B (ICN Biomedicals, Ohio) for fungi was used for susceptibility test of fungal strains, and ciprofloxacin (ICN Biomedicals, Solon, OH, USA) was used for the susceptibility test of bacterial strains. Either the 544ex/590E or BioTek Power Wave XS Plate Reader (Bio-Tek Instruments, Vermont) (A. fumigatus, M. intracellulare) was used to read all organisms before and after the incubation period, using the Polarstar Galaxy Plate Reader (BMG Lab Technologies, Germany). MBC (minimum bactericidal concentration) and MFC (Minimum fungicidal concentration) can be described as the lowermost concentration of the tested substance that kills the organism. e MFC and MBC have been established by taking 5 μL of the individual well and transferred to agar. At

In Vitro Screening of the Antimalarial Effect.
e antimalarial activity was in vitro measured against two strains, Plasmodium falciparum (chloroquine resistant (W2) and chloroquine sensitive (D6)), supplied from the Division of Experimental erapeutics, Walter Reed Army Institute of Research. e antimalarial activity assessment depends on the measurement of LDH (plasmodial lactate dehydrogenase) enzymatic action. A red blood cells (RBCs) suspension infected by both Plasmodium falciparum strains (W2 and D6) (with 2% parasitemia, at 200 ul and two percentage of hematocrit in RPMI 1640 medium supplemented with ten percentage of human serum and sixty µg/ml amikacin) is added to the flat bottom microplates (96-well) containing 10 µl of serially diluted test samples. At 37°C for 72 h, the plate is incubated in a modular incubation chamber flushed with a gas blend of ninety percentage of N 2 , five percent of O 2 , and five percent of CO 2 .
e Makler and Hinrichs procedure was followed to establish the plasmodial LDH action [36]. For half hour, a mixture of 100 µl of Malstat reagent and twenty µL of the incubation mixture were incubated at 25°C. 20 µL of a 1 : 1 mixture of nitroblue tetrazolium (NBT) test, which measures ROS produced by spermatozoa, and phenazine ethosulfate, which is generally used to detect NO reductase activity in concert with ascorbic acid (Sigma), was added. In the dark, the plate was then incubated for an extra 60 minutes. e reaction was stopped by adding 100 µl of five percent of CH 3 COOH, and the plate was measured at 650 nm. e IC50 data were collected via plotting the percentage of growth versus test concentrations. e positive controls in this assay protocol are artemisinin and chloroquine. However, DSMO is used for the placebocontrolled studies.
An in vitro cytotoxicity investigation against mammalian cells SI (selectivity index) determination of the test compounds was conducted. e screening was performed using Vero cells (monkey kidney fibroblasts from the American Type Culture Collection). At a density of twenty-five thousand cells per well, the mammalian cells were scattered and stored at 37°C for 24 h. Variable dilutions were added, and the plates were stored for 48 h. e neutral red assay was followed to determine the number of viable cells, and IC 50 data were then generated from dose-response curves [37] .

In Vitro Assessment of Antileishmanial And Antitrypanosomal Activities.
e in vitro antitrypanosomal and antileishmanial activities of target synthesized derivatives were investigated using stock 2 mg/mL concentration of the target compounds in DMSO. Four serial dilutions of all tested compounds were performed using an incomplete RPMI medium.
In the exponential phase, Leishmania donovani promastigotes (three-day-old culture) were diluted with RPMI medium to 1 × 10 6 cells/ml for promastigote assays. A Leishmania donovani axenic amastigotes three-day-old culture was diluted with the same medium to 2 × 10 6 cells/ml for axenic amastigote assays. In the exponential phase, Trypanasoma brucei (two-day-old culture) was diluted with Iscove's Modified Dulbecco's Medium (IMDM), which is an adapted DME medium containing additional amino acids, sodium pyruvate, high glucose (4,500 mg/L), zwitterionic sulfonic acid buffering agent (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), selenium, and others, to 5 × 10 3 cells/ml for the antitrypanosomal assay. e test compounds with three different serial dilutions were added to the Trypanasoma brucei trypamastigote, Leishmania donovani axenic amastigote, and Leishmania donovani promastigote cultures. All samples have been assessed at three serial dilutions starting from 10 µg/mL to 0.4 µg/mL. At 26°C for 72 hours (37°C for Trypanasoma brucei trypamastigotes and axenic amastigotes), the plates were incubated, and by Alamar Blue assay as previously reported, the growth of the parasites in cultures was established [38][39][40].
Employing a newly developed parasite-rescue and transformation assay, samples were tested versus Leishmania donovani intracellular amastigotes in THP1 cells [41]. In the exponential phase, a four-day-old THP1 cell culture was diluted with a growth medium called the RPMI medium to 2.5 × 10 5 cells/ml.
To the final concentration of 25 ng/ml, PMA was added. In experimental culture plates, the culture treated with PMA was distributed and incubated overnight in a five percentage of CO 2 incubator at 37°C. A serum-free medium was then used to wash the plates by differentiated cells of THP1. For the THP1 toxicity assay, serial dilutions of target derivatives were added upon differentiated cells of THP1. For 48 hours at 37°C, the plates were anaerobically incubated using a five percentage of CO 2 incubator. Alamar Blue assessment was followed for determination of the cell growth, and the IC 90 data were calculated from the dose-response curves.

Molecular Modeling Study.
e protein structure for PfPNP is available at the protein bank database, and the crystal structure (PDB ID: 3PHC) was downloaded from the protein databank repository [42]. PrepWizard (Protein Preparation Wizard) of the Schrӧdinger suite was used to prepare the protein structure. To do so, adding missing hydrogen atoms, relaxing the protein-ligand complex, assigning HBs, and adjusting bond orders [41] were carried out. Prime was applied to fill amino-acid side chains and the missing loops [43]. e cocrystallized ligand coordinates were used to assign the docking receptor grid center.
rough scaling the VDW radius by a partial charge cutoff of 0.25 and factor of 1.0, the potential of the receptor nonpolar parts was softened. For optimum HB shape with docked poses predicted, rotation of the receptor SH and OH groups was permitted using the OPLS3 force field. LigPrep of Schrodinger was performed for ligand preparation [44,45]. To produce potential ligand states at pH 7.4, Epik was used, and during ligand preparation, ligand stereogenic centers were maintained [46]. e glide SP (standard precision) mode was applied to execute virtual compounds docking; consequently, the best five poses were selected [47].

Results and Discussion
3.1. Chemistry. In Scheme 1, synthesis of oxazole derivatives 2a-g, 3, and 4a-d is shown. Simple and straightforward Schiff bases 2a-g were prepared by condensation of the 3-benzoxazolyl aniline 1 with appropriate benzaldehyde under reflux condition in absolute ethanol and with 4-5 drops of glacial acetic acid. Another set of triazine derivatives 4a-d was synthesized through successive nucleophilic substitution of cyanuric chloride with different aryl amines in one pot [48]. Cyanuric chloride was firstly stirred with compound 1 at room temperature for 1 hour in acetone to afford compound 3, followed by heating with the a suitable aromatic amine under reflux in dioxane to yield the triazine derivatives 4a-d.
In Scheme 2, a straightforward reaction of compound 1 with acid chlorides (namely, chloroacetyl chloride and chlorobutyryl chloride) at room temperature in DMF containing potassium carbonate afforded amides 5a,b in quantitative yields is shown. Compound 5a was further derivatized with aromatic amines via nucleophilic substitution of the chlorine atom in compound 5a to yield compounds 6a-d [33,[49][50][51][52]. e structures of all newly prepared derivatives were described and verified with different spectroscopic tools.

In Vitro Antimicrobial Activity.
e antimicrobial effect was evaluated for all the synthesized benzoxazolyl aniline analogs. Antimicrobial actions were assessed against the fungal strains of Cryptococcus neoformans, C. krusei, C. albicans C. glabrata, and Aspergillus fumigatus and the bacterial strains of Mycobacterium intracellulare, Pseudomonas aeruginosa, methicillin-resistant S. aureus (MRSA), Staphylococus aureus, and Escherichia coli. Drug controls (amphotericin for fungi and ciprofloxacin for bacteria) was incorporated in each test as positive controls [35].
All compounds did not exhibit antimicrobial activities, whether for bacteria or fungi, up to 20 µg/mL concentration, except 2-chloroacetamide 5a, with an IC 50 value of 22.3 µM against C. neoformans.
3.3. Antiprotozoal Activity. All the novel prepared derivatives were biologically evaluated in vitro against resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum as potential antimalarial agents [35][36][37]. ree compounds (4a, 5a, and 6d) displayed promising antiprotozoal activity among the other target compounds, and their screening data with the two positive controls (chloroquine and artemisinin) are depicted clearly in Table 1.
Compound 6d showed moderate antileishmanial activity against both L. donovani amastigotes and promastigotes with IC 50 values of 17.9 and 23.7 μM, respectively, along with    3.4. Docking Studies. One important target for antimalarials is PfPNP (P. falciparum purine nucleoside phosphorylase), the key enzyme in purine recycling and salvage. Inhibition of PfPNP results in parasite death through purine starvation, since P. falciparum is a purine auxotroph due to lack of the de novo purine biosynthetic pathway [41]. PfPNP catalyzes inosine phosphorolysis into ribose-1-phosphate and hypoxanthine, the primary purine substrate for the salvage pathway [53]. erefore, we considered PfPNP as a molecular target of the proposed synthesized compounds. e crystal structure of PfPNP is available in the protein data bank (PDB) with PDB ID of 3PHC [54]. e crystal structure of PfPNP showed a homohexamer (in contrast to its human analog, which is a homotrimer [55]), and it is organized as a trimer of dimers, where in each monomer, the active site is completely occupied via the cocrystallized ligand (immucillin) and one sulfate [41]. Two analogous catalytic sites are localized between each of the dimer pairs and are correlated by a noncrystallographic two-fold axis. e virtual docking was performed for the most active compounds (4a and 5a) in order to rationalize the activity of these compounds against malaria [56].
e docking simulations showed that compound 5a was located properly at the binding site. Moreover, the molecular docking of the compounds 5a and 4a into the PfPNP active site showed superior binding affinity of 5a than 4a.
e possible steric hindrance effect of 4,6-diphenylamino-triazine substitution of compound 4a could negatively affect the binding affinity into the active site, which may explain the reduced antimalarial activity for this compound. e benzoxazole moiety was embedded into the hydrophobic pocket delimited by Pro209, Trp212 Tyr160, Met159, Val181, and Met183 and oriented to establish two π-π stacking interactions with Tyr160 and a hydrogen bond with Asp206 ( Figure 3). Mutation studies verified that Asp206 is crucial for catalysis [56].

Conclusions
In summary, simplified novel analogs of naturally occurring antibiotic benzoxazole were prepared and assessed for their antimicrobial, antileishmanial, antimalarial, and antitrypanosomal activities. 3-Benzoxazolyl aniline amidation with chloroacetyl resulted in promising antifungal and antimalarial activities and showed moderate inhibitory activities against Leishmania and Trypanosoma spp. However, the substitution at the amino group with other substituents resulted in diminished inhibitory activities, except the compound 6d that showed good antitrypanosomal activity. Our findings highlight the importance of the chloroacetyl functionalization of benzoxazolyl aniline and provide a good starting point for designing potent and novel antimicrobial and antiprotozoal agents. Further work is being directed toward enhancing the activity and selectivity.

Data Availability
Data are uploaded as a supplementary file.

Conflicts of Interest
e authors declare that they have no conflicts of interest.