Novel benzofuran derivatives: synthesis and antitumor activity

Chemistry

The reaction of 2-acetylbenzofuran with bromine in a mixture of dioxane and ether afforded 1-(benzofuran-2-yl)-2-bromoethanone (1) according to a previously described method [16]. Treatment of 1 with thiosemicarbazide in dioxane gave 2-hydrazinothiazole derivative 2 in excellent yield of 80% [17]. The structure of compound 2 (Scheme 1) was confirmed by analysis of its ¹H NMR spectrum. 4-(1-Benzofuran-2-yl)thiazole-2-amine (4 in Scheme 2) was synthesized in good yield by treatment of thiourea with 2-bromoacetylbenzofuran (1) in boiling ethanol [18].

Scheme 1

Reagents and conditions: (A) NH₂NHCSNH₂, dry dioxane, stirring, rt, 3 h; (B) aldehyde, molecular sieves 4 Å, 1.1 equiv. thioglycolic acid, THF/toluene, Δ, 12–16 h.

Scheme 2

Reagents and conditions: (A) NH₂CSNH₂, absolute ethanol, Δ, 5 h; (B) aldehyde, molecular sieves 4 Å, 1.1 equiv. thioglycolic acid, THF/toluene, Δ, 12–16 h.

The traditional method of the synthesis of thiazolidin-4-ones involves two steps, namely the formation of Schiff base followed by its cyclocondensation with thioglycolic acid [19, 20]. However, one of the major causes of waste in organic reaction chemistry is multi-stage synthesis. Thus, single-step syntheses are very attractive and possess high value in modern chemistry. Therefore, the novel single-step synthesis of compounds 3a–f, 5a–e using a solid catalyst was performed. In this investigation, thiazolidin-4-ones 3a–f, 5a–e were synthesized by molecular sieve – mediated one-pot three-component condensation reaction [21, 22] of an aromatic amine, an aromatic aldehyde and thioglycolic acid. The reaction was carried out in toluene or THF as a solvent. The molecular sieves (zeolite, hydrated metal aluminosilicates) act mainly as a dehydrating agent [23, 24]. The reason for their success in catalysis is related to their high surface area and adsorption capacity.

Both compounds 2 and 4 were heated under reflux with different aromatic aldehydes and thioglycolic acid in THF or toluene as solvent and in the presence of molecular sieve as a catalyst. The thiazolidin-4-ones formation was found to be complete, as determined by TLC analysis, in approximately 12–16 h. Then, the molecular sieve was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was triturated with NaHCO₃ to remove any unreacted thioglycolic acid. It is worth noting that the use of THF as a solvent is preferred due to the observed decrease in reaction time and more facile work-up. The structures of thiazolidin-4-ones 3a–f, 5a–e were assigned on the basis of their analytical and spectral data.

Biological activity

It has been found that a group of rubromycins and their analogs, a class of quinone antibiotics that possess a benzofuran system, strongly inhibit human telomerase enzyme, which leads to telomere shortening, arrest of cell growth and apoptosis [25]. Thus, benzofuran derivatives play an important role in inhibiting telomerase enzyme. All compounds 3a–f and 5a–e were subjected to preliminary screening, using single dose (500 μg/mL), for primary anticancer in vitro assay against HEPG2 (human liver carcinoma cell line), using the sulforhodamine-B (SRB) assay [26] (Table 1). Based on the requirement for cell line screening set by NCI that the percent growth of tumor cells (PG%) is 30% or less in at least one of the cell lines, it may be concluded that all these compounds are active because they approach this value, except 3a which shows a PG% of 41% in HEPG2 cells at a concentration of 500 μg/mL. In addition, further experiments were performed using different concentrations to calculate IC₅₀ values of the most active compounds in comparison with doxorubicin and 5-flurouracil as positive controls. The cytotoxic activity of the tested compounds on HEPG2 were expressed as IC₅₀ (Table 2), where IC₅₀ (μg/mL) is the dose of compound which reduces survival to 50%. The relation between the surviving fraction and drug concentration is plotted to get the survival curve of the tumor cell line (Figure 2).

Figure 2

The standard curves for the most active compounds on HEPG2 liver cell lines compared with standard drugs, doxorubicin (DOX) and 5-flurouracil (5-FU).

Experiments were repeated three times and the results are displayed as mean values. Points are the means and bars are the SD values.

Structure-activity relationship

The structure-activity correlation of the obtained results revealed that the presence of the NH spacer between the thiazolidinone ring and the thiazolylbenzofuran system may be essential for biological activity. Interestingly, the presence of such a spacer in the 3 a–f series plays an important role in increasing antitumor activity (IC₅₀ ≤ 17.8 μg/mL). Compound 3f is the most potent agent with IC₅₀ = 12.4 μg/mL. Furthermore, the results suggest that the different functional groups on the benzene ring, with different hydrophilic properties, also contribute to biological activity. The derivatives 3c and 5c, with two methoxy groups at the ortho and para positions show good antitumor activity. Moreover, the dimethylamino group at the para position of 3f and 5e appears to improve biological activity to some extent. By contrast, compounds 3b and 5b display moderate activity (IC₅₀ ≥ 17.8 μg/mL), indicating the effect of the hydroxy group at the ortho position. In addition, the presence of the hydroxy group in the para position of 3a and 5a dramatically reduces the growth inhibitory potential.

General method for the synthesis 3-(4-(benzofuran-2-yl)thiazol-2-ylamino)-2-(substituted phenyl)thiazolidin-4-ones 3a–f

2-(2-Hydrazinylthiazolyl)benzofuran (0.5 g, 1 mmol) and an aromatic aldehyde (1 mmol) were stirred in THF for 5 min, followed by addition of mercaptoacetic acid (1.1 mmol) and molecular sieves 4 Å. The mixture was stirred under reflux, until TLC showed disappearance of the starting materials. The molecular sieves were removed by filtration. The filtrate was concentrated in vacuo and the residue was triturated with 5% NaHCO₃. The crude product 3a–f was filtered and crystallized from petroleum ether/diethyl ether (2:1).

Synthesis of 4-(benzofuran-2-yl) thiazol-2-amine (4)

A solution of 2-bromoacetylbenzofuran (1.35 g, 0.01 mol) and thiourea (0.4 g, 0.01 mol) in ethanol (10 mL) was heated under reflux for 3 h and then neutralized with 10% K₂CO₃. The separated solid was filtered, dried and crystallized from ethanol/water (9:1) [27]: yield 1.1 g (90%); mp 214°C; ¹H NMR: δ 6.99 (s, 2H, NH₂, D₂O exchangeable), 7.05 (s, 1H, C3′-H), 7.20–7.35 (m, 3H, C5′-H, C6′-H and C4-H), 7.55 (d, 1H, C4′-H, J = 8 Hz); 7.65 (d, 1H, C7′-H, J = 8Hz).

General procedure for the synthesis of 3-(4-(benzofuran-2-yl)thiazol-2-yl)-2-(substituted phenyl)-thiazolidin-4-ones 5a–e

A solution of 2-(2-aminothiazolyl)benzofuran (1.2 g, 1 mmol) and an aromatic aldehyde (1 mmol) in toluene or THF was stirred and treated with mercaptoacetic acid (1.1 mmol) and molecular sieves 4 Å. The mixture was stirred under reflux until TLC showed disappearance of starting materials. The molecular sieves were removed by filtration. The filtrate was concentrated in vacuo and the residue was triturated with 5% NaHCO₃. The crude product 5a–e was filtered and crystallized from petroleum ether/diethyl ether (2:1).

Sulforhodamine-B (SRB) assay of cytotoxicity

The aim of the present study was to analyze the effect of newly synthesized compounds on HEPG2 cells in comparison with doxorubicin and 5-flurouracil as positive controls. Human tumor cell lines were obtained frozen in liquid nitrogen (-180°C) from the American Type Culture Collection, RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO, USA). The medium was prepared and used for culturing and maintenance of the human tumor cell lines. The prepared medium was kept in a refrigerator at 4°C and checked at regular intervals for contamination. Before use, the medium was warmed at 37°C in a water bath and supplemented with penicillin/streptomycin and FBS. Experiments were set up using the SRB assay. Cells were plated in 96-multiwell plates (5 × 104–105cells/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. Each concentration was evaluated three times and each dose was incubated with the cells in three different wells. Monolayer cells were incubated with the compounds for 48 h 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 (Meter Tech. Σ 960, USA). The mean background absorbance was automatically subtracted and a mean value of each drug concentration was calculated. The average values of three calculations are presented as mean±SD (standard deviation), (Table 3). The percentage of cell survival was calculated as follows: survival fraction = OD (treated cells)/OD (control cells).