Design, synthesis and antiviral evaluation of triazole-linked 7-hydroxycoumarin-monoterpene conjugates as inhibitors of RSV replication

Abstract

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infections in babies across the world. Irrespective of progress in the development of RSV vaccines, effective small molecule drugs are still not available on the market. Based on our previous data we designed and synthesized triazole-linked coumarin-monoterpene hybrids and showed that they are indeed effective in inhibiting the RSV replication. The most effective compounds are active against both RSV serotypes, A and B, with IC₅₀ in the low micromolar or submicromolar range of concentrations. These are the most active coumarin derivatives found so far. Compound 45 combining 3,7-dimethyloctane and cyclopentane-annealed coumarin fragments has a selectivity index of 160 for serotype A and 1147 for serotype B. According to the results of the time-of-addition experiments, the conjugates are active at the early stages of the virus cycle. Based on biological evaluation and molecular modeling data, RSV F protein is a possible target.

Fig. 1. Some of the known inhibitors of RSV reproduction.

Fig. 2. Some of the compounds containing monoterpene and coumarin motifs. Values of CC₅₀ and IC₅₀ against RSV A are shown in μM.

Fig. 3. Intermolecular contacts form during the binding of compounds 1 and 10 to the binding site of the F-protein inhibitor.

Fig. 4. Concept of triazole-linked monoterpene–coumarin conjugates.

Scheme 1. Synthesis of 7-hydroxycoumarins 15–18.

Scheme 2. Synthesis of propargyl ethers of 7-hydroxycoumarins 19–22.

Scheme 3. Synthetic routes for the target azides 32–36.

Scheme 4. Synthesis of triazole-linked monoterpene–coumarin hybrids 10, 37–56.

Fig. 5. Results of time of addition assay for 37, 39 and 45. −2 : 24 – the compound was in contact with the cells two hours before infection, as well as after the addition and removal of the virus; −2 : -1 – the compound was added to the cells and washed off before the virus was introduced; −1 : 0 – the compound was added to the cells pre-cooled to 5 °C simultaneously with the virus, and washed along with unbound viral particles at time point 0; 0 : 24 – the compound was added immediately after washing off unbound viral particles; 2 : 24 – the compound was added two hours after the removal of unbound viral particles; 4 : 24 – after 4 hours; 6 : 24 – after 6 hours; 8 : 24 – after 8 hours; 23 : 24 – after 23 hours; VC – virus control, without the addition of the compound; CC – cell control; DC – compound control, not infected, similar to the cell control. The compound was added at time point −2.

Fig. 6. Antiviral activity of compounds 39, 44, 49 and 54 against RSV-A and RSB-B: pIC₅₀ data (reverse decimal logarithm of IC₅₀) are given. The total energy (E, kcal mol⁻¹), calculated by quantum chemical methods (B3LYP-D3/6-311G(d,p)), is represented in blue. Binding energy (ΔG_bind) of the ligand to the binding site of F-protein is shown in green.

Fig. 7. Antiviral activity of compounds 40, 45, 50 and 55 against RSV-A and RSV-B: pIC₅₀ data (reverse decimal logarithm of IC₅₀) are given. Binding energy (ΔG_bind) of the ligand to the binding site of F-protein is shown in green.

Fig. 8. The relationship between the results of molecular docking and the subsequent assessment of binding energy (ΔG_bind) and experimental data in biological experiments. Values of ΔG_bind were estimated using the MM-GBSA method (solvent is water).

Fig. 9. The location of 2 (A), 37 (B), 3 (C) and 54-E (D) in the biding site of F-protein: π–π stacking interactions are shown as blue dotted lines, H-bond – yellow dotted line.

Fig. 10. The location of lead-compound 45 stereoisomers in the binding cavity of F-protein.