In Vitro and In Vivo Antiviral Studies of New Heteroannulated 1,2,3-Triazole Glycosides Targeting the Neuraminidase of Influenza A Viruses

Abstract

There is an urgent need to develop and synthesize new anti-influenza drugs with activity against different strains, resistance to mutations, and suitability for various populations. Herein, we tested in vitro and in vivo the antiviral activity of new 1,2,3-triazole glycosides incorporating benzimidazole, benzooxazole, or benzotriazole cores synthesized by using a click approach. The Cu-catalyzation strategy consisted of 1,3-dipolar cycloaddition of the azidoalkyl derivative of the respective heterocyclic and different glycosyl acetylenes with five or six carbon sugar moieties. The antiviral activity of the synthesized glycosides against wild-type and neuraminidase inhibitor resistant strains of the avian influenza H5N1 and human influenza H1N1 viruses was high in vitro and in mice. Structure-activity relationship studies showed that varying the glycosyl moiety in the synthesized glycosides enhanced antiviral activity. The compound (2R,3R,4S,5R)-2-((1-(Benzo[d]thiazol-2-ylmethyl)-1H-1,2,3-triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Compound 9c) had a 50% inhibitory concentration (IC₅₀) = 2.280 µM and a ligand lipophilic efficiency (LLE) of 6.84. The compound (2R,3R,4S,5R)-2-((1-((1H-Benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate had IC₅₀ = 2.75 µM and LLE = 7.3 after docking analysis with the H5N1 virus neuraminidase. Compound 9c achieved full protection from H1N1 infection and 80% protection from H5N1 in addition to a high binding energy with neuraminidase and was safe in vitro and in vivo. This compound is suitable for further clinical studies as a new neuraminidase inhibitor.

Keywords: antiviral; avian H5N1; glycosides; heterocyclic; human H1N1; neuraminidase inhibitor.

Figure 1

Medicinal chemistry rational and structures of zanamivir, oseltamivir, and selected antiviral candidates.

Figure 2

Preparation of 2-(azidomethyl)benzo[d]imidazole, 2-(azidomethyl)benzo[d]thiazole, and 2-(azidomethyl)benzo[d]oxazole. Synthetic procedures: (i) X = NH: bromoacetic acid, 4N HCl, reflux, 4 h, NH₄OH; X = S or O: chloroacetyl chloride or bromoacetyl bromide, trimethylamine, xylene, 0 °C for 2 h, reflux for 8 h. (ii) ethanol/DMSO/water (8:1:1), NaN₃, 65–75 °C, 7 h.

Figure 3

Synthetic procedure: (i) Propargyl alcohol, BF3.Et2O, CH2Cl2, rt., (ii) CuL3Br, 2-butanol, rt, 48 h.

Figure 4

Cytotoxicity assay of new synthetic compounds, oseltamivir, and zanamivir in MDCK cells. The 50% cytotoxic concentration (CC₅₀) of each tested compound was calculated by using nonlinear regression analysis (GraphPad Prism software).

Figure 5

The 50% Inhibitory concentration (IC₅₀) of new synthetic compounds, oseltamivir, and zanamivir against H5N1_wild, H5N1_V116A, H5N1_N295S, and H1N1 viruses.

Figure 6

Antiviral activity of the new synthetic compounds, oseltamivir, and zanamivir against the H5N1_wild, H5N1_V116A, H5N1_N295S, and H1N1 viruses using a plaque reduction assay.

Figure 7

NA inhibition assay of synthetic compounds versus that of the control oseltamivir and zanamivir against the H5N1_wild, H5N1_V116A, H5N1_N295S, and H1N1 viruses.

Figure 8

Inhibition effect of three promising synthetic compounds (9c, 8b, and 9a) on the propagation of the H5N1_wild, H5N1_V116A, H5N1_N295S, and H1N1 viruses by HA titration with MOI 0.005 after 72 h.

Figure 9

Mortality rate and antiviral efficacy of the new synthetic compounds and NAIs in infected mice. The groups were infected with different viruses and were administered synthetic compounds 8b, 9c, and 9a 4 h post infection and compared with groups that were administered oseltamivir and zanamivir as drug control and compared with a viral control (VC) group, which was infected with a virus but not treated. (A) Study of the antiviral activity after infection with H5N1_wild (B) Study of the antiviral activity after infection with H5N1_N295S (C) Study of the antiviral activity after infection with H5N1_V116A (D) Study efficacy and antiviral activity after infection with H1N1 virus.

Figure 10

TCID₅₀ titration of lung tissue after 3 DPI. (A) 9c was the most promising synthetic compound after infection with H5N1_wild and reduced the propagation of the virus more than oseltamivir and zanamivir. (B) Oseltamivir and zanamivir reduced the propagation of the virus more than synthetic compounds after infection with H5N1V116A. (C) 9a, oseltamivir, and zanamivir similarly reduced the propagation of the virus after infection with H5N1N295S. (D) 9c and 9a reduced the propagation of the virus after infection with H1N1 as much as zanamivir, but oseltamivir did not affect the propagation of the virus in the lung. The significant differences are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns = non-significant.

Figure 11

EID₅₀ titration of nasal washes to detect viral shedding after 5 dpi. (A) Synthetic compounds reduced the viral shedding more than oseltamivir and zanamivir after infection with H5N1_wild. (B) Synthetic compounds 9c and 9a reduced viral shedding from H5N1_V116A-infected cells more than oseltamivir and zanamivir. (C) 9a was the only synthetic compound that reduced the shedding of virus after infection with H5N1_N295S as well as zanamivir and oseltamivir. (D) Synthetic compounds reduced the viral shedding as well as zanamivir after infection with H1N1. The significant differences are indicated as follows: * p < 0.05, ** p < 0.01, and ns = non-significant.

Figure 12

Visual representation of the docked compounds with neuraminidase (PDB ID: 7e6q). (A) The docked ligand (grey) overlay its co-crystalized complex (green); (B) Compound 9b forms two HBs through the nitrogen atoms of its triazole moiety; (C) Compound 8c displays a hydrophobic–hydrophobic interaction; (D) Compound 9c (grey) completely overlays its structural isomer (green); (E) Compound 9a shows a strong HB with Asn:147A and Thr:444A.