Chemicals and reagents
Anthranilic acid, acetic anhydride, aniline, p-toluidine, o-toluidine, acetone, dimethylsulfoxide (DMSO), anhydrous zinc chloride, p-chlorobenzaldehyde, p-nitrobenzaldehyde, p-hydroxybenzaldehyde, chloroform, absolute ethanol, absolute methanol, resazurin sodium salt, anhydrous petroleum ether, distilled water, iodine, Giemsa stain, Tween 80, 1% acacia gum, HCl, and KOH were used in the study. Most of the commercial solvents, chemicals, and reagents were purchased from either Merck or Sigma-Aldrich with the highest purity and used without further purification.
Instruments and apparatuses
Melting points were determined in open capillaries using electro-thermal 9100 melting point apparatus and were uncorrected. The FTIR spectra in nujol were recorded with the SHIMADZU 8400SP FT-IR spectrophotometer (Shimadzu Corporation, Nakagyo-Ku, Kyoto, Japan), and nuclear magnetic resonance (NMR) spectral data were performed on Bruker Avance DMX400 FT-NMR spectrometer (Bruker, Billerica, MA, USA) using tetramethylsilane (TMS) as internal standard. Silica gel TLC plates of 0.25 mm thickness were used in the study.
Experimental animals and strains
Swiss albino male mice of weight 20–32 g and age 6–8 weeks were used for the antimalarial activity and acute toxicity tests. Plasmodium berghei ANKA strain is used to infect the mice for a four-day suppressive test, were obtained from Biomedical Laboratory, Department of Biology, Faculty of Science, AAU. Leishmania donovani isolate used in this study was obtained from the Leishmania Diagnosis and Research Laboratory (LDRL) culture bank, School of Medicine, AAU.
Culture medium and conditions
RPMI-1640, 10% heat-inactivated fetal calf serum (HIFCS), 1% penicillin–streptomycin, and 1% L-glutamine were supplied to make a complete culture medium. The Leishmania donovani isolate was grown first on Novy-MacNeal-Nicolle (NNN) medium and then in tissue-culture flasks containing RPMI 1640 medium supplemented with 10% HIFCS and 1% 100 IU penicillin/mL-100 µg/mL streptomycin solution at 22 ℃ for promastigotes .
For the in vivo antimalarial activity testing, chloroquine phosphate (Ethiopian Pharmaceutical Manufacturer (EPHARM), Addis Ababa, Ethiopia) was used as a reference drug. Miltefosine/hexadecylphosphocholine (AG Scientific, San Diego, CA, USA) and amphotericin B deoxycholate (Fungizone®, ER Squibb, Middlesex, UK) were employed as reference drugs in the in vitro antileishmanial activity testing of the synthesized compounds.
Synthesis of target compounds
The synthesis of 3-aryl-2-styryl substituted-4(3H)-quinazolinones, were achieved using 3-aryl-2-methyl-4(3H)-quinazolinones (3–5) as key intermediates. It involved cyclization, condensation, and hydrolysis reactions (Fig. 1). The details of each reaction and reaction conditions are mentioned in the following sections.
General procedure for the synthesis of 2-methyl-3,1-benzoxazine-4-one
A solution of anthranilic acid (1) (10 g, 0.073 mol) in acetic anhydride (25 mL) was heated under reflux for 1 h. The precipitate formed on cooling was filtered and the excess acetic anhydride was washed with anhydrous petroleum ether, where a solid mass is obtained. The solid mass 2-methyl-3,1-benzoxazine-4-one (2), without purification, was used for the subsequent reaction [10, 40].
General procedure for the synthesis of 3-aryl-2-methyl-4(3H)-quinazolinones
A mixture of 2-methyl-3,1-benzoxazine-4-one (2) (3 g, 0.017 mol) and equimolar amounts of aromatic amines (aniline, o-toluidine, and p-toluidine respectively) was heated under reflux at 190 ℃ for 5 h. The dark sticky mass formed was cooled and recrystallized from ethanol (3–5) .
General procedure for the synthesis of 3-aryl-2-styryl substituted-4(3H)-quinazolinones
To a solution of each 3-aryl-2-methyl substrituted-4(3H)-quinazolinones (3–5) (0.5 g) in acetic anhydride (10 mL), an equimolar amount of the target aromatic aldehyde was added. Anhydrous zinc chloride (10 mg) is added as a catalyst. The reaction mixture is heated under reflux for 8 h, cooled, and poured into ice-cooled water. The solid products formed (6–8 and 10) were filtered, dried, and recrystallized from ethanol [26, 40].
IR (Nujol) (cm−1): 1682 (C = O), 1593 (C = N), 1556 and 1377 (NO2). 1H NMR (CDCl3/CCl4) ppm: 6.56 (d, 1H, J = 15.5 Hz, vinyl-C2 H), 7.36 (d, 2H, J = 8.2 Hz, phenyl-C2,6 H), 7.46 (d, 2H, J = 8.7 Hz, 4-nitrophenyl C2,6 H), 7.52–7.56 (m, 1H, quina-C6 H), 7.60–7.66 (m, 3H, phenyl C3,4,5 H), 7.83–7.85 (m, 2H, quina-C7,8 H), 8.00 (d, J = 15.54 Hz, 1H, vinyl C1 H), 8.18 (d, 2H, J = 8.8 Hz, 4-nitrophenyl C3,5 H), 8.35 (d, J = 8.4 Hz, 1H, quina-C5 H). Anal. calcd. for C22H16N3O3: C, 71.34; H, 4.35; N, 11.35. Found: C, 71.21; H, 4.62; N, 11.58.
IR (Nujol) (cm−1): 1682 (C = O), 1597 (C = N), 1224 (C–Cl). 1H NMR (CDCl3/CCl4) ppm: 2.15 (s, 3H, o-tolyl-CH3), 6.33 (d, 1H, J = 15.5 Hz, vinyl C2 H), 7.23–7.25 (m, 3H, 4-chlorophenyl C3,5 and o-tolyl C3 H), 7.30 (d, 2H, J = 8.6 Hz, 4-chlorophenyl C2,6 H), 7.41–7.54 (m, 4H, o-tolyl C4,5,6 and quina-C6 H), 7.84 (m, 2H, quina-C7,8 H), 7.99 (d, 1H, J = 15.5 Hz, vinyl C1 H), 8.36 (d, 1H, J = 7.9 Hz, quina-C5). Anal. calcd. for C23H17ClN2O: C, 73.69; H, 5.12; N, 7.47; Cl, 9.46. Found: C, 73.26; H, 4.89; N, 7.78; Cl, 9.67.
4-((1E)-2-(3,4-dihydro-4-oxo-3-o-tolylquinazolin-2-yl)vinyl)phenyl acetate (8)
IR (Nujol) (cm−1): 1756 (C = O), 1682 (C = O), 1634 (C = N), 1205 (C–O–C). 1H NMR (CDCl3/CCl4) ppm: 2.15 (s, 3H, phenylacetate-CH3), 2.35 (s, 3H, o-tolyl-CH3), 6.31 (d, 1H, J = 15.5 Hz, vinyl C2 H), 7.06 (d, 2H, J = 8.6 Hz, phenylacetate C3,5 H), 7.25 (d, 1H, J = 7.7 Hz, o-tolyl C3 H), 7.34 (d, 2H, J = 8.6 Hz, phenylacetate C2,6 H), 7.41–7.51 (m, 4H, o-tolyl C4,5,6 and quina-C6 H), 7.823–7.832 (m, 2H, quina-C7,8 H), 8.02 (d, 1H, J = 15.5 Hz, vinyl C1 H), 8.35 (d, 1H, J = 7.9 Hz, quina-C5 H). Anal. calcd. for C25H20N2O3: C, 75.36; H, 5.57; N, 7.03. Found: C, 75.62; H, 5.32; N, 6.88.
4-((1E)-2-(3,4-dihydro-4-oxo-3-p-tolylquinazolin-2-yl)vinyl)-2-methoxyphenyl acetate (10)
IR (Nujol) (cm−1): 1761(C = O), 1682 (C = O), 1614 (C = N), 1260 and 1149 (C–O–C). 1H NMR (CDCl3/CCl4) ppm: 2.3 (s, 3H, phenylacetate CH3), 2.5 (s, 3H, p-tolyl-CH3), 3.8 (s, 3H, OCH3), 6.36 (d, 1H, J = 15.5 Hz, vinyl C2 H), 7.00 (m, 3H, 2-methoxyphenyl C3,5,6 H), 7.22 (d, 2H, J = 8.1 Hz, p-tolyl C3,5 H), 7.41 (d, 2H, J = 8.1 Hz, p-tolyl C2,6 H), 7.51 (m, 1H, quina-C6 H), 7.81 (m, 2H, quina-C7,8 H), 7.93 (d, 1H, J = 15.5 Hz, vinyl C1 H), 8.33 (d, 1H, J = 7.3 Hz, quina-C5 H). Anal. calcd. for C26H22N2O4: C, 72.88; H, 5.65; N, 6.54. Found: C, 73.12; H, 5.35; N, 6.32.
General procedure for the synthesis of 3-aryl-2-(4-deacetylatedstyryl)-4(3H)-quinazolinones
Subsequent treatment of 8–10 with 0.1 M alcoholic KOH (5 mL) (KOH dissolved in ethanol) followed by 0.1 M HCl (6 mL) was done. The resulting 4-hydroxyl bearing target compounds (11–13) was precipitated, filtered, dried, and recrystallized from ethanol [26, 40].
IR (Nujol) (cm−1): 3274 (OH), 1651 (C = O) and 1606 (C = N). 1H NMR (DMSO-d6) ppm: 2.05 (s, 3H, o-tolyl-CH3), 6.05 (d, 1H, J = 15.4 Hz, vinyl C2 H), 6.75 (d, 2H, J = 8.6 Hz, 4-hydroxypheny C3,5 H), 7.20 (d, 2H, J = 8.6 Hz, 4-hydroxyphenyl C2,6 H), 7.38 (d, 1H, J = 7.5 Hz, o-tolyl C3 H), 7.43–7.44 (m, 1H, quina-C6 H), 7.50–7.54 (m, 3H, o-tolyl C4,5,6 H), 7.75–7.77 (d, 1H, J = 8.1 Hz, quina-C8 H), 7.85–7.89 (m, 2H, vinyl C1 and quina-C7 H), 8.14 (d, 1H, J = 9.49 Hz, quina-C5 H), 9.95 (s, 1H, phenolic-OH). Anal. calcd. for C23H18N2O2: C, 77.51; H, 5.66; N, 7.86. Found: C, 77.21; H, 5.78; N, 7.61.
IR (Nujol) (cm−1): 3296 (OH), 1651(C = O) and 1608 (C = N). 1H NMR (DMSO-d6) ppm: 2.44 (s, 3H, p-tolyl CH3), 6.16 (d, 1H, J = 15.4 Hz, vinyl C2 H), 6.76 (d, 2H, J = 8.4 Hz, 4-hydroxyphenyl C3,5 H), 7.22 (d, 2H, J = 8.4 Hz, 4-hydroxyphenyl C2.6 H), 7.32 (d, 2H, J = 8.06 Hz, p-tolyl C3,5 H), 7.42 (d, 2H, J = 7.9 Hz, p-tolyl C2,6 H), 7.476–7.513 (m, 1H, quina-C6 H), 7.746 (d, 1H, J = 8.2 Hz, quina-C8), 7.78–7.86 (m, 2H, quina-C7 and vinyl C1 H), 8.11 (d, 1H, J = 8.0 Hz, quina-C5 H), 9.98 (s, 1H, phenolic-OH). Anal. calcd. for C23H18N2O2: C, 77.51; H, 5.66; N, 7.86. Found: C, 77.21; H, 5.26; N, 7.48.
IR (Nujol) (cm−1): 3423 (OH), 1683 (C = O), 1634 (C = N), 1211and 1148 (C–O–C). 1H NMR (CDCl3/CCl4) ppm: 2.5 (s, 3H, p-tolyl CH3), 3.8 (s, 3H, OCH3), 5.95 (s, 1H, phenolic-OH), 6.3 (d, 1H, J = 15.4 Hz, vinyl C2 H), 6.82–6.92 (m, 3H, 4-hydroxy-3-methoxyphenyl C3,5,6 H), 7.22 (d, 2H, J = 8.3 Hz, p-tolyl C3,5 H), 7.40 (d, 2H, J = 8.0 Hz, p-tolyl C2,6 H), 7.48 (m,1H, quina-C6 H), 7.78–7.79 (m, 2H, quina-C7,8 H), 7.92 (d, 1H, J = 15.4 Hz, vinyl C2 H), 8.30–8.32 (d, 1H, J = 7.9 Hz, quina-C5 H). Anal. calcd. for C24H20N2O3: C, 74.59, H, 5.74; N, 7.23. Found: C, 74.43; H, 5.48; N, 6.92.
Preparation of stock and working solutions
Stock solutions of 10 mg/mL of the synthesized compounds were prepared by dissolving each compound in DMSO for the antileishmanial activity testing. Stock solutions were diluted using complete RPMI to obtain aliquots of 10 µg/mL. Then, three-fold serial dilution with complete RPMI gave the final six working concentrations (10, 3.33, 1.11, 0.37, 0.12, and 0.04 µg/mL) of each of the synthesized compounds. Amphotericin B deoxycholate and miltefosine, which were used as positive controls for the comparison of the antileishmanial activities of the test compounds, were also made in three-fold serial dilutions. All the prepared drugs were stored at − 20 °C and retrieved only during use .
In vivo antimalarial activity test
The antimalarial activities of the target compounds were assessed by the four-day standard suppressive test against mice infected with Plasmodium berghei [42, 43]. The mice were acclimatized to the experimental conditions in the animal house of Biomedical Laboratory, Addis Ababa University, Faculty of Science, Department of Biology, for seven days prior to the in vivo antimalarial activity testing. In due course, the mice were subjected to live in standard cages with a pelleted diet and water. To trigger a steadily rising infection in mice, blood infected with Plasmodium berghei ANKA strain (approximately 20–30% level of parasitemia) was collected from a donor mice using a syringe supplemented with 0.5% trisodium citrate and diluted in physiological saline to 107 parasitized erythrocytes per mL. Then, 0.2 mL of inoculum (supposed to have about 2 × 107 parasites) was injected into each mouse through the intraperitoneal route to achieve the desired level of infection within a short period of time . After 2 h of post-infection, the mice were carefully weighed using a digital balance and randomly assigned into nine groups where each group contain five mice. Of which, mice assigned as a negative control (Group 1) subjected to the formulation composed of 7% Tween 80, and 3% ethanol in distilled water (2 mL/100 g). In addition, mice which belong to the positive control (Group 2) was treated with 25 mg/kg/day (0.04846 mmol/kg/day) of chloroquine phosphate (CQ). The remaining groups (3–9) were subjected to oral treatment with equimolar amounts of the respective target compounds for four consecutive days. Finally, a blood smear was taken from the tail of the mice in the 5th day (24 h after the last dose), air-dried, fixed with absolute methanol and treated with 6% Giemsa stain. The mean parasitemia level of each group (control and treatment groups) was computed microscopically by counting the number of erythrocytes in four fields (⁓100 erythrocytes per field). In the end, the antimalarial activities of the target compounds were expressed in terms of blood parasitemia, percent suppression and mean survival time of mice in comparison with the negative- and positive-control groups .
In vitro antileishmanial activity test
Prior to the in vitro antipromastigote testing, serial concentrations of the target compounds were prepared by implementing three-fold dilutions. Then, in a 96-well microtiter plate, 100 µl of each of the three-fold serial dilutions of the target compounds were added in triplicate wells. Then 100 µl of Leishmania donovani suspension containing 3.0 × 106 promastigotes per milliliter were added in duplicates. In this experiment, the media and DMSO served as a negative control. Moreover, the concentration of DMSO was maintained below 1% of the target compound preparation to avoid the potential growth inhibition and interference effect of DMSO in the entire antipromastigote assay . Properly labelled plates which belong to the negative-control group, positive-control groups (treated with miltefosine and amphotericin B deoxycholate) and target compounds were then kept in a humidified atmosphere at 22 ℃ under 5% CO2 for 68. To determine the viability Leishmania donovani isolates after the respective treatment, 10 µL of fluorochrome AlamarBlue® solution prepared by dissolving 12.5 mg resazurin in 100 mL of distilled water was added to each well and incubated for 4 h. Then, the fluorescence intensity of each well was determined using Victor3 Multilabel Counter (PerkinElmer, Waltham, MA, USA), at 530 nm and 590 nm, excitation and emission wavelengths, respectively . The IC50 values of the negative-control, positive-control groups and test-groups were determined from sigmoidal dose-response curves using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA).
Molecular docking study
Molecular docking study was carried out for compound 6 which elicited pronounced antileishmanial activity, using AutoDock Vina by retrieving the three-dimensional structure of Lm-PTR1 from the Protein Data Bank (PDB ID: 2bfm). The complex with Trimethoprim forming by chain A of the Lm-PTR1 heterodimer was employed in the modeling study. Ligands were sketched in a manner which minimized energy and the protein was formulated via the Discovery studio suite (V5.1). The Python script (prepare receptor4.py) offered by the MGLTools package (version 1.5.4) was followed to convert protein files to PDBQT format for docking using AutoDock Vina (version 1.1.2). The efficiency of the search algorithm was maintained at its default setting. The grid box docking dimensions were -3.629 Å × 41.782 Å × 66.219 Å, with a spacing of 1 Å to deal with all the possible conformations of the docked molecule. All graphical representations in Fig. 3 were extracted using Pymol.
In vivo acute toxicity test
The preliminary safety profiles of target compounds (6, 8, and 10) with pronounced bioactivities were investigated using male Swiss albino mice (⁓20 g each) . For this purpose, the mice were randomly allocated into six groups each containing six mice. The first five groups of mice (Group 1–5) received each target compound solubilized using 1% gum acacia at a dose of 10, 50, 100, 200 and 300 mg/kg, respectively. The mice in last group (group 6, served as a negative control) were orally treated with the solvent used to dissolve the target compounds (1% gum acacia) at a maximum dose of 1 mL/100 gm of body weight . Each mouse was then monitored for gross changes such as salivation, loss of appetite, lacrimation, hair erection, diarrhea, convulsions, death, and other signs of overt toxicity.
The protocols that involving experimental animals were assessed and approved by the Institutional Ethics Review Committee, School of Pharmacy, Addis Ababa University. In addition, the study reported in accordance with the Animal Research Reporting of in vivo Experiments (ARRIVE) guidelines  and were handled according to the Guide for the Care and Use of Laboratory Animals (https://olaw.nih.gov/sites/default/files/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf).
The antimalarial activities of the synthesized compounds were computed as mean ± standard deviation and one-way ANOVA was used to test the statistical significance for the suppressive test using Origin 6.0 software. Data on % suppression, % parasitemia and mean survival time was analyzed using Microsoft office excel 2007. Moreover, all the data were analyzed at a 95% confidence interval. The IC50 values for in vitro promastigotes assay of target compounds were determined from sigmoidal dose-response curves using computer software GraphPad Prism 5.0.