Synthesis and biological evaluation of new 3-(4-substituted phenyl)aminoquinoxaline derivatives as anticancer agents

Chemistry

This study was initiated by the synthesis of 2-(4-acetylphenylamino)-3-quinoxaline derivatives (3a,b) through the reaction of 2,3-dichloroquioxaline derivatives with an equimolar amount of 4-aminoacetophenone according to the literature method [30].

The synthesis of enones 4a,b was achieved by condensation of the methyl ketones 3a,b with furfuraldehyde in the presence of sodium hydroxide [31]. Meanwhile, the hydrazones 5a,b were synthesized by the reaction of methyl ketones 3a,b with salicylic acid hydrazide (Scheme 1) [32].

Enones 4a,b are an important synthon for the construction of variety of heterocycles [33–35]. In this work, the Michael addition reaction of compounds 4a,b with malononitrile in the presence of sodium alkoxide afforded cyano alkoxypyridines 6a–d. A similar cyclocondensation of 4a,b in the presence of ammonium acetate gave aminopyridines 7a,b. The treatment of enones 4a,b with ethyl cyanoacetate in the presence of ammonium acetate gave products 8a,b (Scheme 2). The structures 6a,d, 7a,b, and 8a,b were confirmed by elemental analyses and spectral data.

Reaction of compounds 4a,b with hydroxylamine in boiling ethanolic solution of potassium hydroxide gave the corresponding oxazole derivatives 9a,b. Meanwhile, enones 4a,b were condensed with hydrazine hydrate and phenyl hydrazine to give the corresponding pyrazolines 10a–d. Also, dihydropyrimidines 11a,b were synthesized by the reaction of 4a,b with urea in ethanolic HCl solution (Scheme 3).

Molecular modeling

It has been suggested that compound Q22 is an anticancer agent as CDK inhibitor using docking procedure with the enzyme 1KE8 [26]. The enzyme 1KE8 (Figure 4) was downloaded from the Protein Data Bank (PDB) with the active ligand 4-{[(2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]amino}-N-(1,3-thiazol-2-yl)benzenesulfonamide [36] (Figure 5).

Figure 4

Structure of the enzyme 1KE8.

Figure 5

Complex of 1KE8 with the benzenesulfonamide active ligand.

Docking studies have suggested that the most important amino acids residues observed for the complex of this active ligand with 1KE8 are asparagine 132, lysine 129, aspartate 127, glutaminate 12, and threonine 14. This complex is shown in Figure 5. Our docking studies showed that derivatives 4–11 can be docked in the same binding site.

Compound Q22 forms one hydrogen bond with leucine 83 amino acid in the enzyme 1KE8 active side. The enone 4b, which is a structural analogue of Q22, also forms one hydrogen bond with the same amino acid leucine 83, as shown in Figure 6. Importantly, the ligand-enzyme binding energy is decreased to -130.79 kJ/mol from -83.16 kJ/mol from the complex of Q22.

Figure 6

Complex of 4b with the 1KE8 binding side.

Similar complexes were generated for other ligands 6–9 (not shown). For example, compound 11a forms six hydrogen bonds with the key amino acid residues in the enzyme active side. One hydrogen bond is between the 3-nitrogen atom of the dihydropyrimidine ring and the oxygen atom of the leucine 83 amino acid. The second bond connects the oxygen atom (2-oxo in the dihydropyrimidine ring) and the nitrogen atom of the leucine 83 amino acid. The third bond is between the furan oxygen atom and the nitrogen atom of the aspartate 86 amino acid. The fourth interaction is formed between the anilino nitrogen atom and the oxygen atom of aspartate 145 amino acid. The last two non-bonding interactions are formed by the bifurcated hydrogen bonds between the oxoquinoxaline oxygen atom and two nitrogen atoms of asparagine 132 lysine 129 amino acids. In comparison to the complex of 4b, the ligand-enzyme binding energy is decreased to -145.07 kJ/mol for 11a. The greater binding affinity of 11a to the 1KE8-binding side (Figure 7) is nicely paralleled by the greater biological activity of 11a in comparison to that of 4b. Thus, cyclization of enone moiety in compound 4a to the dihydropyrimidine ring of 11a results in an increased activity. Similar results were obtained for the remaining compounds (Figures 8, 9 and Table 1).

Figure 7

Docking of 11a in 1KE8 binding side.

Figure 8

Docking of 9b in 1KE8 binding side.

Figure 9

Docking of 7b in 1KE8 binding side.

Biological activity

Many natural quinoxaline derivatives are antitumor antibiotics [37–40]. All synthetic quinoxalines 4–11 were screened in vitro, using single dose (500 μg/mL), against HEPG₂ (human liver carcinoma cell line), using the sulforhodamine-B (SRB) assay [41] (Table 1). Based on the requirement set by NCI that the growth percent of tumor cells (PG%) is 30% or less for active agents lines, it may be concluded that most of these compounds are active because their activities approach this value. The exception is 4a, which shows a PG% of 43% in HEPG₂ cells at a concentration of 500 μg/mL.

Chemistry

Melting points were recorded using Fisher-Johns apparatus and are uncorrected. Elemental analyses were performed at the Microanalytical Center, Cairo University, Egypt. IR spectra were recorded on Mattason 500 FT-IR spectrometer in KBr pellets. The ¹H NMR spectra (400 MHz) and ¹³C NMR spectra (100 MHz) were recorded in DMSO-d₆ on a Bruker 400 spectrometer at Georgia State University (Atlanta, GA, USA). Mass spectra were obtained on a JOEL JMS-600H spectrometer at Cairo University.

Synthesis of 1-{4-[3-chloroquinoxalin-2-yl)amino] phenyl}ethan-1-one (3b)

A mixture of 2,3-dichloro-6-methylquinoxaline (2b, 2.13 g, 0.01 mol) and 4-aminoacetophenone (2.02 g, 0.015 mol) in absolute ethanol (10 mL) was heated under reflux for 6 h and then cooled. The resultant precipitate was collected by filtration, dried, and crystallized from acetone to give 3b: yield (2.43 g, 78%); mp 228–230°C; IR: 3311 (NH), 3026 (CH), 1678 cm^-1 (CO); ¹HNMR: δ 2.30 (s, 3H), 2.60 (s, 3H), 7.39–8.01 (m, 7H), 9.00 (s, 1H); ¹³C NMR: δ 21.5, 28.2, 113.1, 127.5, 130.7, 132.7, 134.4, 137.2, 138.3, 139.9, 140.5, 142.1, 147.1, 165.1, 181.1. Anal. Calcd for C₁₇H₁₄ClN₃O (311.77): C, 65.43; H, 4.49; N, 13.47. Found: C, 65.47; H, 4.46; N, 13.5.

General method for the synthesis of 3-({4[3-(furan-2-yl)-1-oxo-prop-2-en-1-yl]phenyl}amino)-7-substituted quinoxalin-2(1H)-one derivatives 4a,b

A mixture of 3a or 3b (0.01 mol) and furfuraldehyde (0.96 g, 0.01 mol) in ethanolic sodium hydroxide solution (10%, 25 mL) was stirred at room temperature for 10 h. The precipitated solid was collected by filtration, dried, and crystallized from ethanol.

General procedure for the synthesis of 1-({[4-(3-chloro-6-substituted quinoxalin-2-yl)amino]phenyl}ethylidene)-2-hydroxybenzohydrazide derivatives 5a,b

A mixture of ketone 3a or 3b (0.001 mol) and salicylic acid hydrazide (0.152 g, 0.001 mol) in acetic acid (5 mL) was heated under reflux for 10 h. After cooling, the resultant precipitate was collected by filtration, dried, and crystallized from glacial acetic acid.

General procedure for the synthesis of 2-alkoxy-4-(furan-2-yl)-6-{[4-(3-oxo-6-substituted-3,4dihydroquinoxalin-2-yl)amino]phenyl}-pyridine-3-carbonitrile derivatives 6a–d

A cooled freshly prepared solution of sodium alkoxide (0.001 mol) [0.023 g sodium metal 0.001 mol in 50 mL absolute methanol for compounds (6a,b) or absolute ethanol for compounds (6c,d)] was treated with malononitrile (0.066 g, 0.001 mol) and then with compound 4a or 4b (0.001 mol) at 60°C for 10 h. The precipitated solid was collected by filtration, dried, and crystallized from ethanol.

General procedure for the synthesis of 2-amino-4-(furan-2-yl)-6-{[4-(3-oxo-6-substitutd-3,4-dihydroquinoxalin-2-yl)amino]phenyl}pyridine-3-carbonitrile derivatives 7a,b

A mixture of compound 4a or 4b (0.001 mol), malononitrile (0.066 g, 0.001 mol), and ammonium acetate (0.616 g, 0.008 mol) in absolute ethanol (25 mL) was heated under reflux for 8 h. The resultant solid was collected by filtration, dried, and crystallized from ethanol.

General method for the synthesis of 4-(furan-2-yl)-2-oxo-6-{4-[(3-oxo-6-substituted-3,4-dihydroquinoxalin-2-yl)amino]phenyl}pyridine-3-carbonitrile derivatives 8a,b

A mixture of compound 4a or 4b (0.001 mol), ethyl cyanoacetate (0.112 g, 0.001 mol), and ammonium acetate (0.616 g, 0.008 mol) in absolute ethanol (25 mL) was heated under reflux for 8 h. The resultant solid was collected by filtration, dried, and crystallized from ethanol.

General procedure for the synthesis of 3-({4-[5-(furan-2-yl)-4,5-dihydroisox azol-3-yl]phenyl}amino)-7-substituted quinoxalin-2(1H)-one derivatives 9a,b

A mixture of compound 4a or 4b (0.001 mol), hydroxylamine hydrochloride (0.14 g, 0.002 mol), and sodium hydroxide solution (0.5 g in 2 mL water) in absolute ethanol (25 mL) was heated under reflux for 8 h. After addition of ice-cold water, the resultant solid was collected by filtration, dried, and crystallized from ethanol.

General method for the synthesis of 3-({4-[5-(furan-2-yl)-4,5-dihydro-1-substituted-pyrazol-3-yl]phenyl}amino)-7-substituted quinoxalin-2(1H)-one derivatives 10a–d

A mixture of compound 4a or 4b (0.001 mol) and hydrazine hydrate (98%) or phenyl hydrazine (0.001 mol) in ethanol or acetic acid, respectively (5 mL), was heated under reflux for 8 h. The resultant solid was collected by filtration, washed several times with hot ethanol and dried.

General method for the synthesis of 3-({4-[6-(furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]phenyl}amino)-7-substituted quinoxalin-2(1H)-one derivatives 11a,b

A mixture of compound 4a or 4b (0.001 mol), urea (0.1 g, 0.001 mol), and concentrated hydrochloric acid (2 mL) in ethanol (25 mL) was heated under reflux for 8 h. After cooling, the precipitated solid was collected by filtration, dried, and crystallized from ethanol.

Molecular modeling

The Molegro Virtual Docker (MVD) program was used to perform docking. The protein-ligand interaction energies of the examined compounds were calculated using the Pose Organizer option in the MVD program [42] for five different orientations.

The structure of enzyme 1KE8 was downloaded from the PDB. The docking results are shown in Table 1.

Biological activity

SRB assay of cytotoxicity was used to analyze the effect of the synthesized compounds on the HEPG₂ human tumor cell line. The tumor cells were obtained frozen in liquid nitrogen (-180°C) from the American type culture collection, RPMI-1640 medium (Sigma Chemicals, St. Louis, MO, USA). Monolayer cells were incubated with the compounds for 48 h before use; the medium was warmed at 37°C in a water bath and supplemented with penicillin/streptomycin and FBS. Cells were planted in 96-multiwell plates (5×10⁴–10⁵ cells/well) for 24 h before treatment with the compounds to allow attachment of cells to the wall of the plate. Different concentrations of the compounds tested (0, 5, 12.5, 25 and 50 μg/mL) were added to the cell monolayer at 37°C and in an atmosphere of 5% CO₂. Control cells were treated with vehicle alone. Cultures were then fixed with trichloroacetic acid and stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mm unbuffered Tris base [tris(hydroxymethyl)aminomethane] for determination of optical density (OD). The OD of each well was measured spectrophotometrically at 564 nm with an ELIZA microplate reader. The mean background value of each drug concentration was calculated. The percentage of cell survival was calculated as follows: survival fraction=OD (treated cells)/OD (control cells).