Quaternary ammonium salts based on (-)-borneol as effective inhibitors of influenza virus

Abstract

A series of compounds containing a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment were evaluated for their antiviral activity against influenza A virus strain A/Puerto Rico/8/34 (H1N1) in vitro. The most potent antiviral compound proved to be a quaternary ammonium salt based on (-)-borneol, 10a. In in vitro experiments, compound 10a inhibited influenza A viruses (H1, H1pdm09, and H3 subtypes), with an IC₅₀ value of 2.4-16.8 µM (depending on the virus), and demonstrated low toxicity (CC₅₀ = 1311 µM). Mechanism-of-action studies for compound 10a revealed it to be most effective when added at the early stages of the viral life cycle. In direct haemolysis inhibition tests, compound 10a was shown to decrease the membrane-disrupting activity of influenza A virus strain A/Puerto Rico/8/34. According to molecular modelling results, the lead compound 10a can bind to different sites in the stem region of the viral hemagglutinin.

Fig. 1

Anti-influenza drugs approved for clinical use to treat influenza virus infection

Fig. 2

(+)-Camphor and (-)-borneol derivatives with potent activity against influenza viruses A(H1N1)pdm09. SI is the selectivity index.

Scheme 1

Reagents and conditions: (i) N,N-dimethylethylenediamine or N,N-dimethylpropane-1,3-diamine, ZnCl₂, reflux under solvent-free conditions; (ii) CH₃I, CH₃CN, reflux

Scheme 2

Synthesis of the (-)-borneol derivatives 8a-c, 9a-c, 10a and c and 11a and b. Reagents and conditions: (i) corresponding chloroacetyl chloride, Et₃N, CH₂Cl₂, r.t.; (ii) the corresponding amine, Et₃N, CH₂Cl₂, r.t.; CH₃I, CH₃CN, reflux

Fig. 3

ORTEP representations of compound 10a. The displacement ellipsoids are drawn at a probability of 30%. Only one of the two independent molecules is shown.

Fig. 4

Time-of-addition activity of compound 10a against influenza A virus PR8 (H1N1). MDCK cells were infected with influenza virus, and the salt 10a was added and removed at the indicated time points, where 0 corresponds to the moment when the cells were infected. The infectious activity of viral progeny was tested by further titration on MDCK cells.

Fig. 5

Haemolysis-inhibiting activity of compound 10a against influenza A virus HA. The compound was mixed with 128 hemagglutinating units of influenza virus, incubated at room temperature for 30 min, mixed with 0.75% chicken erythrocytes, and incubated at +4°C. After incubation with MES buffer (0.1 M MES, 0.15 M NaCl, 0.9 mM CaCl₂, 0.5 mM MgCl₂, pH 5.0) and sedimentation of erythrocytes, the optical density in the wells was measured at 405 nm. The haemolysis-inhibiting activity of compound 10a was calculated by comparison to the haemolysis activity of influenza A virus without additives.

Fig. 6

Location of lead compound 10a in the binding sites of different HAs. The secondary structure of an HA subunit corresponding to PDB code 1RU7 (PR8 (H1N1)) is shown in blue, that of PDB code 3EYM (Aichi (H3N2)) is shown in orange, and that of PDB code 3LZG (Cal (H1N1)pdm09) is shown in green. H-bonds and salt-bridges are presented as yellow and pink dotted lines, respectively, and π-cation interactions are shown as green dotted lines.

Fig. 7

The result of protein alignment procedure. The secondary structure of an HA subunit corresponding to PDB code 1RU7 (PR8 (H1N1)) is shown in blue, that of PDB code 3EYM (Aichi (H3N2)) is shown in orange, and that of PDB code 3LZG (Cal (H1N1)pdm09) is shown in green. Amino acid sequence differences are highlighted in red.