Pharmacophore-based virtual screening, synthesis, biological evaluation, and molecular docking study of novel pyrrolizines bearing urea/thiourea moieties with potential cytotoxicity and CDK inhibitory activities

Abstract

In the current study, virtual screening of a small library of 1302 pyrrolizines bearing urea/thiourea moieties was performed. The top-scoring hits were synthesised and evaluated for their cytotoxicity against three cancer (MCF-7, A2780, and HT29) and one normal (MRC-5) cell lines. The results of the MTT assay revealed potent cytotoxic activities for most of the new compounds (IC₅₀ = 0.16-34.13 μM). The drug-likeness study revealed that all the new compounds conform to Lipinski's rule. Mechanistic studies of compounds 18 b, 19a, and 20a revealed the induction of apoptosis and cell cycle arrest at the G₁ phase in MCF-7 cells. The three compounds also displayed potent inhibitory activity against CDK-2 (IC₅₀ = 25.53-115.30 nM). Moreover, the docking study revealed a nice fitting of compound 19a into the active sites of CDK-2/6/9. These preliminary results suggested that compound 19a could serve as a promising scaffold in the discovery of new potent anticancer agents.

Keywords: CDK-2; Pyrrolizine; apoptosis; cell cycle; cytotoxicity; urea derivatives.

Graphical abstract

Figure 1.

Multi-CDK inhibitors 1–5 bearing similar pharmacophoric features.

Figure 2.

Diary urea derivatives 6–9 with their inhibitory activities against CDKs.

Figure 3.

Rational design of scaffold A and general structure of the compound library.

Figure 4.

Compound 3 (LZ9) bound to CDK-2 (pdb 2VTP); (A) pharmacophoric features of compound 3; (B) pharmacophoric features selected to generate the 3D pharmacophore model used in the virtual screening.

Figure 5.

Virtual screening and structural modifications of scaffold A.

Scheme 1.

Reagents and conditions: (a) (CH₃)₂SO₄, benzene, CH₂(CN)₂; (b) ClCH₂COCl, g. acetic acid, CH₂COONa; (c) acetone, K₂CO₃, reflux, 24 h.

Scheme 2.

Reagents and conditions: (d) hexyl isocyanate, TEA, DCM, stir, rt, 12 h; (e) appropriate isocyanate, TEA, DCM, stir, rt, 12 h; (f) appropriate isocyanate/isothiocyanate, TEA, DCM, stir, rt, 12 h.

Figure 6.

SAR of cytotoxicity and selectivity of the new compounds against MCF-7, A2780, and HT29 cells.

Figure 7.

Annexin V phases of MCF-7 treated with compound 18b, 19a, and 20a at 0, 1, 5, and 10 µM (24 h, x-axis: Annexin V; y-axis: PI). C1: necrotic cells; C2: late apoptosis; C3: live cells; C4: early apoptosis. Data shown is mean % cell number ± SD (n = 3). Experiment was repeated x3 times.

Figure 8.

Flow cytometry bar chart showing the effect of compounds 18b, 19a, and 20a at 0, 1, 5, and 10 μM on cell cycle distribution of MCF-7 cells after treatment for 24 h, values represent the mean ± SD (n = 3), experiment was repeated ×3.

Figure 9.

Binding mode and interactions of compound 19a into CDK-2 (pdb: 2VTP)): (A) 3D binding mode of compound 19a into the active site of CDK-2, the native ligand LZ9 shown as yellow sticks, receptor shown as hydrogen-bond surface; (B) 2D binding mode of compound 19a into CDK-2 showing three conventional hydrogen bonds, one pi-cation, and several hydrophobic interactions, hydrogen atoms were omitted for clarity.

Figure 10.

Binding modes and interactions of compounds 18b/20a into CDK-2 (pdb: 2VTP)): A) 3 D binding mode of compound 18b into CDK-2, the native ligand LZ9 shown as yellow sticks, receptor shown as hydrogen-bond surface; B) 3 D binding mode of compound 20a into CDK-2, the native ligand LZ9 shown as yellow sticks; C) 2 D binding mode of compound 18b into CDK-2 showing three conventional hydrogen bonds and several hydrophobic interactions; (C) 2D binding mode of compound 20a into CDK-2 showing four conventional hydrogen bonds and one pi-anion, and several hydrophobic interactions, hydrogen atoms were omitted for clarity.

Figure 11.

3D binding modes, hydrogen bonding (shown as green dotted lines), and electrostatic interactions (shown as orang dotted lines) of compounds 18b, 19a, and 20a (shown as sticks) into CDK-6 (pdb: 2EUF) and CDK-9 (pdb: 3TNH): (A) compound 18b into CDK-6; (B) compound 19a into CDK-6; (C) compound 20a into CDK-6; (D) compound 18b into CDK-9; (E) compound 19a into CDK-9; (F) compound 20a into CDK-9, amino acid residues shown as lines coloured by element, hydrogen atoms were omitted for clarity.