Synthesis and Biological Evaluation of Benzo [4,5]- and Naphtho[2',1':4,5]imidazo[1,2-c]pyrimidinone Derivatives

Abstract

Azacarbazoles have attracted significant interest due to their valuable properties, such as anti-pathogenic and antitumor activity. In this study, a series of structurally related tricyclic benzo[4,5]- and tertacyclic naphtho[2',1':4,5]imidazo[1,2-c]pyrimidinone derivatives with one or two positively charged tethers were synthesized and evaluated for anti-proliferative activity. Lead tetracyclic derivative 5b with two amino-bearing arms inhibited the metabolic activity of A549 lung adenocarcinoma cells with a CC₅₀ value of 3.6 μM, with remarkable selectivity (SI = 17.3) over VA13 immortalized fibroblasts. Cell-cycle assays revealed that 5b triggers G2/M arrest without signs of apoptosis. A study of its interaction with various DNA G4s and duplexes followed by dual luciferase and intercalator displacement assays suggests that intercalation, rather than the modulation of G4-regulated oncogene expression, might contribute to the observed activity. Finally, a water-soluble salt of 5b was shown to cause no acute toxic effects, changes in mice behavior, or any decrease in body weight after a 72 h treatment at concentrations up to 20 mg/kg. Thus, 5b is a promising candidate for studies in vivo; however, further investigations are needed to elucidate its molecular target(s).

Keywords: anti-proliferative activity; azacarbazoles; cytostatic agent; imidazopyrimidinone.

Figure 1

Examples of derivatives of γ-carboline (A,E) and carbazole (B–D). An example of synthesis of the adduct of 2′-deoxycytidine with 1,4-benzoquinone (F). Blue scaffolds are γ-carboline and carbazole.

Scheme 1

Synthesis of benzo- and naphtho-imidazopyrimidinone derivatives. Reagents and conditions: (a) 1,4-benzoquinone, 0.1 M sodium acetate buffer (pH 4.5), 37 °C, 24 h for 2a (60%) or 1,4-naphthoquinone, 0.1 M sodium acetate buffer (pH 4.5)/C₂H₅OH (2:1, v:v), 50 °C, 7d for 2b (21%); (b) ethane-1,2-diamine, CH₃OH, 50°, 48 h for 3a (83%), 3b (80%), 5a (91%), 5b (90%); (c) DBU, methyl 2-bromoacetate, CH₂Cl₂, rt, 3h for 4a (43%) and 4b (48%); (d) CH₂O, NaCNBH₃, AcOH, MeOH, 0 °C→rt, 2h for 6a (38%) and 6b (33%) and 1H-pyrazole-1-carboxamidine hydrochloride, DIPEA, DMSO, 60 °C 3h for 7a (47%) and 7b (50%).

Figure 2

(A) Dose–response dependencies of cytotoxicity for HEK293T, MCF7′, A549, and VA13 cell lines after a 72 h treatment with 5b. (B) Changes in cell cycle distribution of lung cancer cells A549 after a 20 h treatment with 5b, paclitaxel, and 92504.

Figure 3

Heatmap of the influence of the compounds on the thermal stability of DNA targets. The concentrations of the compounds and the targets were 1 and 20 µM, respectively. For all targets except VEGF, buffer 1 (20 mM sodium phosphate, pH 7.4, 10 mM KCl) was used. For VEGF, buffer 2 (5 mM sodium phosphate, pH 7.4, 25 mM LiCl) was used.

Figure 4

Heatmap of changes in the G4-stabilizing effects of the compounds in the presence of duplex DNA. ND—not determined. The concentrations of the compounds, ds26, and the targets were 10 µM, 10 µM, and 0.5 µM, respectively. Buffer 1 (20 mM sodium phosphate, pH 7.4, 10 mM KCl) was used for all targets, and buffer 2 (5 mM sodium phosphate, pH 7.4, 25 mM LiCl) was used for VEGF..

Figure 5

Heatmap of the dissociation constants K_d of G4:derivative complexes according to MST. ND—not determined.