N-Substituted Pyrrole-Based Heterocycles as Broad-Spectrum Filoviral Entry Inhibitors

Abstract

Since the largest and most fatal Ebola virus epidemic during 2014-2016, there have been several consecutive filoviral outbreaks in recent years, including those in 2021, 2022, and 2023. Ongoing outbreak prevalence and limited FDA-approved filoviral therapeutics emphasize the need for novel small molecule treatments. Here, we showcase the structure-activity relationship development of N-substituted pyrrole-based heterocycles and their potent, submicromolar entry inhibition against diverse filoviruses in a target-based pseudovirus assay. Inhibitor antiviral activity was validated using replication-competent Ebola, Sudan, and Marburg viruses. Mutational analysis was used to map the targeted region within the Ebola virus glycoprotein. Antiviral counter-screen and phospholipidosis assays were performed to demonstrate the reduced off-target activity of these filoviral entry inhibitors. Favorable antiviral potency, selectivity, and drug-like properties of the N-substituted pyrrole-based heterocycles support their potential as broad-spectrum antifiloviral treatments.

Figure 1:

Crystal structure of EBOV GP trimer (PDB 5JQ7), composed of GP₁ (light blue) and GP₂ (dark blue) subunits within each monomer. The toremifene binding pocket at the internal fusion loop region is indicated in yellow.

Figure 2:

Structure-activity relationship diagram of the hit N-substituted furopyrrole CBS1111.

Figure 3:

Toremifene (yellow) bound to EBOV GP (a) and docking model for compound 49 (pink) at the internal fusion loop region (b). Residues implicated for ligand binding are displayed in blue. Modeling was performed using PDB 5JQ7.

Figure 4:

Pseudoviral activity of derivatives 11 (a), 49 (b), and 58 (c) using wildtype and mutated EBOV GP. Results suggest the internal fusion loop region as the derivative binding site. Pseudoviral activity was determined in A549 cells. EC₅₀ curves were calculated using the four-parameter nonlinear logistic regression analysis in GraphPad Prism.

Figure 5:

Time-of-addition assay of 58 against pMARV (a) and pEBOV (b) suggests the N-substituted heterocycles inhibit viral entry using a similar mechanism as toremifene. Pseudotyped MARV or EBOV were incubated in A549 cells at 4°C for 1 hour at t=−1. After incubation, the temperature was shifted to 37°C to trigger viral entry. AZT, toremifene, and 58 were introduced at different time points to assess inhibition efficacy. Data are represented as mean from six replicates ± standard deviation.

Figure 6:

Pseudoviral H5N1 Influenza and VSV activity of derivatives 5 (a), 11 (b), and 49 (c). Pseudoviral activity was determined in A549 cells.

Scheme 1:

Synthetic procedure for the N-substituted furopyrrole (5-16, 23-35) and thienopyrrole (17-22) series. Reagents and conditions: (a) NaH, DMF 0 °C to RT; (b) NaOH, THF/MeOH/H₂O; (c) DIPEA/HATU, DMF 0 °C to RT.

Scheme 2:

Synthetic procedure for the N-substituted indole (46-59, 60-63) and pyrrole (64) series. Reagents and conditions: (a) NaH, DMF 0 °C to RT; (b) NaOH, THF/MeOH/H₂O; (c) DIPEA/HATU, DMF 0 °C to RT. A1, A10, and A12 refer to the corresponding amines displayed in Scheme 1.

Scheme 3:

Synthetic procedure for the benzyl aniline derivative (69). Reagents and conditions: (a) NaH, DMF 0 °C to RT; (b) NaOAc, EtOH reflux; (c) NaOH, THF/MeOH/H₂O; (d) DIPEA/HATU, DMF 0 °C to RT.