Design and Optimization of Quinazoline Derivatives: New Non-nucleoside Inhibitors of Bovine Viral Diarrhea Virus

Abstract

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus (Flaviviridae). In spite of the availability of vaccines, the virus is still causing substantial financial losses to the livestock industry. In this context, the use of antiviral agents could be an alternative strategy to control and reduce viral infections. The viral RNA-dependent RNA polymerase (RdRp) is essential for the replication of the viral genome and constitutes an attractive target for the identification of antiviral compounds. In a previous work, we have identified potential molecules that dock into an allosteric binding pocket of BVDV RdRp via a structure-based virtual screening approach. One of them, N-(2-morpholinoethyl)-2-phenylquinazolin-4-amine [1, 50% effective concentration (EC₅₀) = 9.7 ± 0.5 μM], was selected to perform different chemical modifications. Among 24 derivatives synthesized, eight of them showed considerable antiviral activity. Molecular modeling of the most active compounds showed that they bind to a pocket located in the fingers and thumb domains in BVDV RdRp, which is different from that identified for other non-nucleoside inhibitors (NNIs) such as thiosemicarbazone (TSC). We selected compound 2-[4-(2-phenylquinazolin-4-yl)piperazin-1-yl]ethanol (1.9; EC₅₀ = 1.7 ± 0.4 μM) for further analysis. Compound 1.9 was found to inhibit the in vitro replication of TSC-resistant BVDV variants, which carry the N264D mutation in the RdRp. In addition, 1.9 presented adequate solubility in different media and a high-stability profile in murine and bovine plasma.

Keywords: BVDV inhibitors; RdRp protein; molecular dynamics; pharmacokinetics in vitro properties; quinazoline derivatives.

Figure 1

Cytotoxicity and anti-BVDV activity of active compound 1 derivatives. (A) Cytotoxicity. Sub-confluent MDBK cell monolayers grown in 96-well plates were treated with 2-fold serial dilutions of each compound in IM. Mock-treated cells were added as control. Cells were incubated and allowed to proliferate for 3 days at 37°C and 5% CO₂. Cell viability was then determined by the MTS/PMS method. (B) Antiviral activity. Sub-confluent MDBK cell monolayers grown in 96-well plates were infected with BVDV NADL (MOI = 0.01) and treated with different concentrations of the compounds. Mock-infected cells and mock-treated infected cells were included as controls in each plate. After 3 days at 37°C and 5% CO₂, cell viability was determined by the MTS/PMS method, and the percentage of the viral CPE inhibition was calculated.

Scheme 1

General procedure for the synthesis of quinazoline derivatives.

Figure 2

Summary of SAR analysis of quinazoline analogs.

Figure 3

Predicted interactions of active compounds 1.9 (pink) and 1.21 (orange) within the allosteric site of BVDV RdRp protein obtained by MD simulations.

Figure 4

Antiviral activity of 1.9 against TSC BVDV-resistant mutants. The anti-BVDV assay was performed as stated in Materials and Methods. For infection, wtBVDV NADL and two different TSC-resistant BVDV NADLs, BVDV R1 (NS5B A392E) and BVDV R2 (NS5B N264D), were used. Compound 1.9 was used at its maximum non-cytotoxic concentration (12.5 μM). As positive control, TSC was used at 34 μM. Mock-infected cells and non-treated infected cells (NTC) were included as controls in each plate. After 3 days, cell viability was determined by the MTS/PMS method, and the percentage of the viral CPE relative to the corresponding NTC was calculated. *p < 0.05 relative to NTC.

Figure 5

(A) Predicted pose of compound 1.9 (pink) within the allosteric site of the RdRp protein obtained by MD simulations. A392 and N264 are in CPK style. (B) Insert shows compound 1.9 located between the fingers and thumb domains of BVDV RdRp. Some amino acids were omitted for clarity.

Figure 6

Experimental stability of compound 1.9. (A) Stability profile of compound 1.9 in SGF, SIF, and PBS. (B) Stability profile in mouse/murine and bovine plasma samples of 1.9 and control drugs (enalapril and SIH) in mouse/murine and bovine plasma samples, respectively. Plots represent the mean percentage of compound remaining against time with the error bars representing the standard deviation of three independent experiments.