Synthesis, characterization and antimalarial evaluation of new β-benzoylstyrene derivatives of acridine

2.1 Synthesis

3.1 Measurement of inhibition of P. falciparum growth in culture

In this study chloroquine sensitive 3D7 and Chloroquine resistant Dd2 strains provided by MR4/ATCC (Manassas, USA) were used in in vitro culture. Parasite strains were cultivated by the method of Trager and Jensen (1976) with minor modifications (Trager and Jensen, 1976). Cultures were maintained in fresh O⁺ human erythrocytes at 4% hematocrit in complete medium [RPMI 1640 (supplemented with 25 mM HEPES and l-Glutamine) with 0.2% sodium bicarbonate 0.5% Albumax, 45 mg/L hypoxanthine and 50 mg/L gentamicin] at 37 °C under reduced O₂ (gas mixture 5% O₂, 5% CO₂, and 90% N₂). Stock solutions of Chloroquine (CQ) were prepared in water (miliQ grade) and test compounds were dissolved in DMSO. All stocks were then diluted with Complete culture medium to achieve the required concentrations (In all cases the final concentration contained 0.4% DMSO, which was found to be non-toxic to the parasite). CQ and test compounds were then placed in 96-well flat-bottomed tissue culture grade plates to yield triplicate wells with drug concentrations ranging from 0 to 10⁻⁴ M in a final well volume of 100 μl for primary screening. Chloroquine was used as a positive control in all experiments. Parasite culture was synchronized at the ring stage with 5% sorbitol. Synchronized culture was aliquoted to drug containing a 96-well plate at 2% hematocrit and 1% parasitemia. After 48 h of incubation under standard culture conditions, plates were harvested by the addition of SYBR Green I containing lysis buffer (100 μl) and read by the SYBR Green I fluorescence-based method (Smilkstein et al. 2004) using a 96-well fluorescence plate reader (Victor, Perkin Elmer), with excitation and emission wavelengths at 497 and 520 nm, respectively. The fluorescence readings were plotted against drug concentration, and IC₅₀ values obtained by visual matching of the drug concentration giving 50% inhibition of growth.

3.2 Measurement of cytotoxic activity against mammalian cell lines in culture

Animal cell line (Hela) was used to determine drug toxicity by using MTT assay for mammalian cell viability assay as described by Mosmann 1983. Hela cells cultured in complete RPMI (cRPMI) containing 10% fetal bovine serum, 0.2% sodium bicarbonate, 50 mg/L gentamycin. Briefly, Cells (10⁴ cells/200 μl/well) were seeded into 96-well flat-bottomed tissue-culture plates in a complete culture medium. Drug solutions were added after overnight seeding and incubated for 24 h in a humidified atmosphere at 37 °C and 5% CO₂. DMSO (final concentration 10%) was added as +ve control. An aliquot of a stock solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL in 1X phosphate-buffered saline) was added at 20 μl per well, and incubated for another 3 h. After spinning the plate at 1500 RPM for 5 min, supernatant was removed and 100 μl of the stop agent DMSO was added to the well to dissolve the formazan crystals. Formation of formazon, an index of growth was read at 570 nm by a 96-well plate reader (Versa Max) and IC₅₀ values were determined by analysis of dose–response curves. Therapeutic index was calculated as IC₅₀ Mammalian cell/IC₅₀ Pf3D7.

4.1 Chemistry

The synthetic pathway for β-benzoylstyrene derivatives of acridine (4a–r) is illustrated in Scheme 1. Compound 4-chloro-2-(4-methoxyanilino) benzoic acid (1) was prepared by performing Ullmann condensation between 2,4-dichlorobenzoic acid and p-anisidine. Subsequently the refluxing of 4-Chloro-2-(4-methoxyanilino) benzoic acid in phosphorus oxychloride resulted in the formation of 6,9-dichloro-2-methoxyacridine (2) (Csuk, et al., 2004). Condensation of 6,9-dichloro-2-methoxyacridine and 4-aminoacetophenone afforded 1-(4-(6-chloro-2-methoxyacridin-9-ylamino) phenyl) ethanone (3) (Aly and Abadi, 2004). Compound 1-(4-(6-chloro-2-methoxy acridin-9-ylamino)phenyl) ethanone was reacted with a series of substituted aromatic aldehydes to obtain β-benzoylstyrene derivatives of acridine (4a–r) (Table 1). All compounds synthesized as above have been characterized by spectral data (IR, ¹H NMR, Mass).

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Scheme 1

Reagents and conditions: (i) K₂CO₃/Cu/Amyl alcohol, p-anisidine 5 h under reflux (ii) POCl₃ reflux for 6 h (iii) HCl, Ethanol, 110 °C for 8 h, (iv) Sodium hydroxide, Methanol, rt, 8–12.

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4.2 Antimalarial activity

All the synthesized compounds were evaluated for their antimalarial activity against CQ-sensitive (3D7) as well as CQ-resistant (Dd2) strains of P. falciparum through in vitro red blood cell based culture using SYBR-Green-I assay (Smilkstein et al., 2004). The results of antimalarial potencies are summarized in Table 1. Variations of different substituents on the aromatic ring of β-benzoylstyrene derivatives of acridine have been explored to ascertain the structure–activity relationship among the synthesized compounds of the series. Among the eighteen evaluated compounds of the series against CQ-sensitive (3D7), three compounds (4c, 4d, 4l) were most potent and showed antimalarial IC₅₀ values in the range of 0.30–0.52 μM. Six compounds (4a, 4b, 4f, 4j, 4k, and 4m) displayed antimalarial activity in the range 0.7–1.5 μM. Nine compounds (4e, 4g, 4h, 4i, 4n, 4o, 4p, 4q and 4r) exhibited the IC₅₀ values ranging from 1.6–4.8 μM. However, activity results clearly suggest that among the monomethoxy substituted compounds (4d, 4g, 4l), the 4-substituted methoxy compound 4l is the most potent (IC₅₀ = 0.3 μM). Indeed compound 4l is six times more active than its 3-methoxy counterpart (compound 4g, IC₅₀ = 1.8 μM) whereas the 2-methoxy substitution (4d, IC₅₀: 0.52 μM) results in a slight decrease in activity. Additional methoxy group at position-3 (4i, 3,4-dimethoxy) leads to a significant fall in activity (IC₅₀ = 4.0 μM).

Isomeric compounds 4c (2-nitro, IC₅₀: 0.35 μM), 4f (3-nitro, IC₅₀: 1.3 μM) and 4k (4-nitro, IC₅₀: 0.7 μM) suggest that positional isomerism of the Nitro group on the aromatic ring of β-benzoylstyrene can cause subtle changes in potency. It was interesting to see over fivefold increase in activity ongoing from compound 4e (2-hydroxy, IC₅₀: 4.5 μM) to compound 4m (4-hydroxy, IC₅₀: 0.8 μM). The 3, 4-dihyroxy compound 5 (IC₅₀: 2 μM) although less active than compound 4m (4-hydroxy, IC₅₀: 0.8 μM) is nevertheless more active than compound 4e (2-hydroxy, IC₅₀: 4.5 μM). With reference to the unsubstituted compound 4a (IC₅₀: 0.75 μM) substitution with methoxy (compound 4d, IC₅₀: 0.52 μM) or nitro (compound 4c, IC₅₀: 0.35 μM) appeared to potentiate antimalarial activity while substitutions by hydroxyl (compound 4e, IC₅₀: 4.5 μM) or chlorine (compound 4b, IC₅₀: 1.5 μM) brought about a reduction in potency.

The two substitutions at position-3 (methoxy compound 4g, Nitro compound 4f) indicate that these substitutions lead to marginal decrease in potency in comparison with the unsubstituted compound 4a. The compounds 4c and 4l were found to be nearly equipotent against both Chloroquine sensitive 3D7 and Chloroquine resistant Dd2 strains, giving resistance indices in the range of 0.88–1. Interestingly compounds 4b, 4d, 4f, 4g, 4h, 4j, 4n, 4o and 4r were found to be more potent against the Chloroquine resistant Dd2 strain than the Chloroquine sensitive 3D7 strain resulting in resistance indices in the range of 0.15–0.7.

All the synthesized compounds were further evaluated for Cytotoxicity by MTT assay (Mosmann, 1983) against HeLa cell line. Interestingly, as shown in Table 1, all the compounds evaluated showed good selectivity indices in the range of 20–520. The compounds 4c, 4d, 4l were the most potent against malarial parasites also exhibited high selectivity indices in the range of 80–520.