4.1 Chemistry
4.1.1 General
Reagents and solvents were purchased from commercial suppliers and used as received. Dry solvents were obtained according to standard procedures. Column chromatography was performed on Macherey-Nagel 60–200 µm silica gel. All the target compounds reported in this publication were of at least 98% purity. The synthesis of derivatives 1, 2 [10] and 8a-c, 9a-c [18] we described previously. Compounds 6-7, 10a,c and 11a,b have not been described previously in the literature. 1H and 13C NMR spectra were recorded on Bruker AV-300 (300.13 and 75.47 MHz, respectively), AV400 (400.13 and 100.78 MHz, respectively) and DRX 500 (500.13 and 125.76 MHz, respectively) spectrometers in CDCl3; chemical shifts δ, in ppm relative to residual [δ(CHCl3) 7.24, δ(CDCl3) 76.90 ppm]. The atom numbering in the compounds is given for assigning the signals in the NMR spectra and does not match the standard nomenclature of compounds. Elemental analysis was carried out using a Euro EA 3000 C, H, N, S-analyser. Analysis of Br was carried out by the mercurimetric titration method. Analysis of I was carried out by the iodometric titration method. X-ray data were collected at room temperature using a Bruker Kappa Apex II CCD diffractometer with graphite monochromated MoKα radiation (λ = 0.71073 Å) with φ, ω-scan method. The data were corrected for absorption using a multi-scan method with the SAINT program. The structure was solved by direct methods using SHELXS97, and refinement was carried out by a full-matrix least-squares technique, using SHELXL97. Anisotropic displacement parameters were included for all non-hydrogen atoms. All H atoms were positioned geometrically and treated as riding on their parent C atoms.
4.1.2 General procedure for the target camphor derivatives 6 and 7.
A solution of compounds 1 or 2 (4 mmol) and anhydrous CH3CN (10 mL) was treated with an excess of iodomethane and heated in a bath at 70–75°C for 6 h. The solvent was removed at reduced pressure. The resulting precipitate was purified via silica gel column chromatography (CHCl3/MeOH eluent, (100:0→0:100)).
N,N,N-Trimethyl-2-((E)-((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)amino)ethanaminium iodide (6).
Yield: 46%; mp: 195–197°C; 1H NMR (400 MHz, CDCl3, J Hz) δ ppm: 0.67 (3H, s, Me-9), 0.84 (3H, s, Me-8), 0.88 (3H, s, Me-10), 1.17-1.27 (2H, m, H-4endo, H-5endo), 1.59-1.68 (1H, m, H-5exo), 1.77-1.86 (2H, m, H-2endo, H-4exo), 1.94-1.99 (1H, m, H-3), 2.36-2.46 (1H, m, H-2exo), 3.52 (9H, s, Me-13, Me-14, Me-15), 3.64-3.75 (2H, m, H-12), 3.85-3.95 (2H, m, H-11). 13C NMR (75 MHz, CDCl3) δ: 186.36 s (C-1), 66.41 t (C-12), 54.61 q (Me-13, Me-14, Me-15), 53.90 s (C-6), 46.97 s (C-7), 46.47 t (C-11), 43.35 d (C-3), 35.61 t (C-2), 31.54 t (C-5), 26.71 t (C-4), 19.20 q (Me-9), 18.39 q (Me-10), 10.81 q (Me-8). Anal. Calcd for C15H29IN2 C, 49.45; H, 8.02; I, 34.83; N, 7.69 %. Found, %: С 48.54; Н 8.10; N 7.64; I 34.57.
N,N,N-Trimethyl-3-((E)-((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)amino)propan-1-aminium iodide (7)
Yield, 76%; mp: 231°C; 1H NMR (400 MHz, CDCl3) δ: 0.71 (3H, s, Me-9), 0.88 (3H, s, Me-8), 1.00 (3H, s, Me-10), 1.23-1.39 (2H, m, H-4endo, H-5endo), 1.64-1.73 (1H, m, H-5exo), 1.76-1.85 (1H, m, H-4exo), 1.98-2.02 (1H, m, H-3), 2.11 (1H, d, 2J=19.6, H-2endo), 2.20-2.29 (2H, m, H-12), 2.54-2.62 (1H, m, H-2exo), 3.41 (9H, s, Me-14, Me-15, Me-16), 3.41-3.47 (2H, m, H-13), 3.71-3.77 (2H, m, H-11). 13C NMR (75 MHz, CDCl3) δ: 191.13 s (C-1), 64.86 t (C-13), 55.35 t (C-11), 53.93 q (Me-14, Me-15, Me-16), 48.13 s (C-6), 46.96 s (C-7), 43.46 d (C-3), 36.75 t (C-2), 31.86 t (C-5), 26.56 t (C-4), 23.94 t (C-12), 19.58 q (Me-9), 18.52 q (Me-10), 11.40 q (Me-8). Anal. Calcd for C16H31IN2 C, 50.79; H, 8.26; I, 33.54; N, 7.40%. Found, %: С 49.68; Н 8.19; N 7.32; I 33.82.
4.1.3 Synthesis of(-)-borneol derivatives 10a, 10c and 11a-b.
N,N,N-Trimethyl-2-oxo-2-((1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy)ethanaminium iodide (10a)
A solution of 8a (4 mmol) and anhydrous CH3CN (10 mL) was treated with an excess of iodomethane and heated in a bath at 70–75°C for 6 h. The solvent was removed at reduced pressure. The crude product was purified by recrystallisation from CH3CN. Yield: 62%; mp:242°C; 1H NMR (400 MHz, DMSO-d6, J Hz) δ ppm: 0.83 (3H, s, Me-9), 0.87 (3H, s, Me-8), 0.89 (3H, s, Me-10), 1.04(1H, dd, 2J = 13.7, J2endo,1exo=3.5, H-2endo), 1.17-1.25 (1H, m, H-4endo), 1.27-1.37 (1H, m, H-5exo), 1.67-1.77 (2H, m, H-3, H-4exo), 1.79-1.87 (1H, m, H-5endo), 2.27-2.37 (1H, m, H-2exo), 3.24 (9H, s, Me-13, Me-14, Me-15), 4.50 (1H, AB-d, J1,2=16.7 Hz, H-12), 4.55 (1H, AB-d, J2,1=16.7 Hz, H-12), 4.95 (1H, m, H-1exo). 13C NMR (125 MHz, DMSO-d6) δ: 165.28 s (C-11), 81.75 d (C-1), 62.89 t (C-12), 53.46 q (Me-13, Me-14, Me-15), 48.86 s (C-6), 47.79 s (C-7), 44.32 d (C-3), 36.03 t (C-2), 27.69 t (C-4), 26.85 t (C-5), 19.69 q (Me-9), 18.77 q (Me-10), 13.61 q (Me-8). Anal. Calcd for C15H28INO2 C, 47.25; H, 7.40; I, 33.28; N, 3.67 %. Found, %: С 47.19; Н 7.26; I 33.21; N 3.73.
Crystal data for 10a
C15H28INO2, M = 381.28, monoclinic, space group P21, a = 12.2791(5), b = 7.1686(3), c = 20.9901(9) Å, β = 101.030(2)°, V = 1813.50(13) Å3, Z = 4, Dcalc = 1.396 mg•m-3, μ = 1.766 mm-1. Data collection yielded 45735 reflections with θ < 30.2° resulting in 10595 unique, averaged reflections, 7365 with I > 2σ(I). Full-matrix least-squares refinement led to a final R = 0.0440, wR2 = 0.1375, GOF = 0.936 for I > 2σ(I). CCDC 1813408 contains supplementary crystallographic data for the structure. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html.
N,N,N-Trimethyl-4-oxo-4-((1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy)butan-1-aminium iodide (10c)
The synthesis of compound 10c was performed in analogy to the synthesis compound 10a. Yield: 43%; mp:171.5°C; 1H NMR (400 MHz, CDCl3, J Hz) δ ppm: 0.76 (3H, s, Me-9), 0.81 (3H, s, Me-8), 0.83 (3H, s, Me-10), 0.90 (1H, dd, 2J=13.7, J2endo,1exo=3.5, H-2endo), 1.12-1.28 (2H, m, H-4endo, H-5exo), 1.60-1.73 (2H, m, H-3, H-4exo), 1.76-1.86 (1H, m, H-5endo), 2.00-2.10 (2H, m, H-13), 2.22-2.32 (1H, m, H-2exo), 2.49 (2H, t, J=6.7 Hz, H-12), 3.42 (9H, s, Me-15, Me-16, Me-17), 3.66-3.75 (2H, m, H-14), 4.76-4.84 (1H, m, H-1exo). 13C NMR (75 MHz, CDCl3, J Hz) δ ppm: 171.9 s (C-11), 80.4 d (C-1), 65.4 t (C-14), 53.4 q (Me-15, Me-16, Me-17), 48.3 s (C-6), 47.3 s (C-7), 44.3 d (C-3), 36.2 t (C-2), 29.4 t (C-12), 27.4 t (C-4), 26.5 t (C-5), 19.2 q (Me-9), 18.3 q (Me-10), 18.0 t (C-13), 13.1 q (Me-8). Anal. Calcd for C17H32INO2 C, 49.88; H, 7.88; N, 3.42 %. Found, %: С 49.87; Н 7.71; N 3.20.
N,N-Diethyl-N-methyl-2-oxo-2-((1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy)ethanaminium iodide (11a)
A solution of 9a (0.2 mmol) and anhydrous CH3CN (5 mL) was treated with an excess of iodomethane and refluxed for 6 h. The solvent was removed at reduced pressure. The crude product was purified by recrystallisation from CH3CN. Yield: 51%; mp:159.2-159.3°C; 1H NMR (400 MHz, CDCl3, J Hz) δ ppm: 0.80 (3H, s, Me-9), 0.83 (3H, s, Me-8), 0.85 (3H, s, Me-10), 1.01 (1H, dd, 2J=13.7, J2endo,1exo=3.5, H-2endo), 1.17-1.35 (2H, m, H-4endo, H-5exo), 1.42 (6H, t, J=7.2, Me-15, Me-16), 1.62-1.76 (2H, m, H-3, H-4exo), 1.78-1.90 (1H, m, H-5endo), 2.25-2.38 (1H, m, H-2exo), 3.48 (3H, s, Me-17), 3.75-3.95 (4H, m, H-13, H-14), 4.55 (2H,AB, H-12), 4.91-5.00 (1H, m, H-1exo).13C NMR (100 MHz, CDCl3, J Hz) δ ppm: 164.3 s (C-11), 83.2 d (C-1), 58.7 t (C-12), 57.8 t (C-13, C-14), 48.8 s (C-6), 48.6 q (Me-17), 47.8 s (C-7), 44.4 d (C-3), 36.2 t (C-2), 27.6 t (C-4), 26.8 t (C-5), 19.4 q (Me-9), 18.5 q (Me-10), 13.5 q (Me-8), 8.3 q (Me-15, Me-16). Anal. Calcd for C17H32INO2 С 49.87; Н 7.71; N 3.20 %. Found, %: С 50.29; Н 8.08; N 3.14.
N,N-Diethyl-N-methyl-3-oxo-3-((1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy)propan-1-aminium iodide (11b)
The synthesis of compound 11b was performed in analogy to the synthesis compound 11a. Yield: 53%; mp:147.3-147.7°C; 1H NMR (400 MHz, CDCl3, J Hz) δ ppm: 0.81 (3H, s, Me-9), 0.84 (3H, s, Me-8), 0.86 (3H, s, Me-10), 1.00 (1H, dd, 2J=13.7, J2endo,1exo=3.5, H-2endo), 1.21-1.34 (2H, m, H-4endo, H-5exo), 1.42 (6H, t, J=7.2, Me-16, Me-17), 1.65-1.76 (2H, m, H-3, H-4exo), 1.82-1.91 (1H, m, H-5endo), 2.26-2.36 (1H, m, H-2exo), 2.96 (2H, t, J=7.1 Hz, H-12), 3.29 (3H, s, Me-18), 3.59-3.71 (2H, m, H-14, H-15), 3.75 (2H, t, J=7.1, H-13), 4.85-4.90 (1H, m, H-1exo). 13C NMR (100 MHz, CDCl3, J Hz) δ ppm: 169.5 s (C-11), 81.9 d (C-1), 57.3 t (C-14, C-15), 56.0 t (C-13), 48.6 s (C-6), 48.2 q (Me-18), 47.8 s (C-7), 44.5 d (C-3), 36.4 t (C-2), 28.1 t (C-12), 27.7 t (C-4), 26.9 t (C-5), 19.5 q (Me-9), 18.6 q (Me-10), 13.5 q (Me-8), 8.3 q (Me-16, Me-17). Anal. Calcd for C18H34INO2 С 51.06; Н 8.09; N 3.42 %. Found, %: С 50.50; Н 8.22; N 3.17.
4.2 Biological assays
4.2.1 Cells and viruses
Influenza virus A/Puerto Rico/8/34 (H1N1) was obtained from the collection of viruses of St Petersburg Pasteur Institute, Russia, and used in the study. Prior to the experiment, the virus was propagated in the allantoic cavity of 10-12 days-old chicken embryos for 48 hr at 36ºC. Infectious titer of the virus was determined in MDCK cells (ATCC # CCL-34) in 96-wells plates in alpha-MEM medium (Biolot, St.-Petersburg, Russia).
4.2.2 Cytotoxicity assay
The microtetrazolium test (MTT) was used to study the cytotoxicity of the compounds. Briefly, a series of threefold dilutions of each compound in MEM were prepared. The MDCK cells were incubated for 48 h at 36°C in 5% CO2 in the presence of the dissolved substances. The cells were washed twice with phosphate-buffered saline (PBS), and a solution of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (ICN Biochemicals Inc. Aurora, Ohio) (0.5 μg/mL) in PBS was added to the wells. After 1 h incubation, the wells were washed, and the formazan residue was dissolved in DMSO (0.1 mL per well). The optical density in the wells was then measured on a Victor2 1440 multifunctional reader (Perkin Elmer, Finland) at a wavelength of 535 nm and plotted against the concentration of compounds. Each concentration was tested triplicate. The 50% cytotoxic concentration (CC50) of each compound was calculated from the data obtained.
4.2.3 In vitro antiviral activity
The compounds were dissolved in 0.1 mL DMSO to prepare stock solutions, and final solutions (1000.0–4.0 μM) were prepared by adding MEM with 1 μg/mL trypsin. The compounds were incubated with MDCK cells for 1 h at 36°C. Each concentration of the compounds was tested in triplicate. The cell culture was then infected with the influenza virus A/Puerto Rico/8/34 (H1N1) (MOI 0.01) for 24 h at 36°C in the presence of 5% CO2. A virus titre in the supernatant was determined by a haemagglutination test after cultivation of the virus in the MDCK cells for 48 h at 36°C in the presence of 5% CO2. Rimantadine, amantadine, deitiforin, and ribavirin were used as reference drugs. For calculations, the virus titre was expressed as per cent of the titre in control wells without compounds. The 50% inhibiting concentrations (IC50) and the selectivity index (SI, the ratio of CTC50 to IC50) were calculated from the obtained data.
4.2.4 Time-of-addition experiments
To determine which stage of the viral life cycle is affected by the compound, cells were seeded into 24-wells plates and incubated with the influenza virus A/Puerto Rico/8/34 (H1N1) (m.o.i. 10) for 1 h at 4°C. After washing the non-absorbed virions for 5 min with MEM, the plates were incubated for 8 h at 36°C at 5% CO2. The starting point of this incubation was referred to as 0 hours. The cells were treated with compound 11 for the following time periods: -2 to -1 (before infecting); -1 to 0 (simultaneously to absorption); 0 to 2; 2 to 4; 4 to 6; 6 to 8; and -2 to 8 hours. In each case after incubation the compound was removed, and cells were washed for 5 min with MEM. After 8 hours of growth, the infectious titer of the virus was determined in the culture medium, as described above.
4.2.5 Hemagglutination inhibition assayand haemolysis assay
In order to assess the ability of compounds to interfere directly with HA receptor binding, we performed hemagglutination inhibition test. Two-fold dilutions of influenza A/Puerto Rico/8/34 virus-containing culture medium (1:8 to 1:256) were mixed with leading compound at a range of concentrations and incubated for 1 hour at 36°C at 5% CO2 followed by adding equal volume of 1% chicken erythrocytes. After incubation for 1 hour at 20°C the results were checked visually. Anti-hemagglutinin activity was evaluated by the ability of specimens to prevent virus-driven hemagglutination.
The membrane-disrupting activity of viral hemagglutinin was measured according to Maeda and Ohnishi [22] with slight modifications. Briefly, chicken erythrocytes were washed twice with PBS and resuspended to make a 0.75% (vol./vol.) suspension in PBS, which was stored at 4°C until use. One hundred microliters of compound diluted in PBS to appropriate concentrations was mixed with an equal volume of the influenza virus (128 hemagglutinating units per 0.1 mL), or PBS for negative control. After incubating the virus-compound mixture at room temperature for 30 min, the mixture was mixed with 300 µL of 0.75% chicken erythrocytes. Mixture was incubated for 1 hour at +4°C for absorption of virions on erythrocytes. 500 µL of MES buffer (0.1M MES, 0.15M NaCl, 0.9mM CaCl2, 0.5mM MgCl2, pH 5.0) was added, mixed and incubated for 1 hour at 37°C for HA acidification and hemolysis. To separate non-lysed erythrocytes, tubes were centrifuged at the end of incubation at 1,200 rpm for 6 min. After sedimentation of erythrocytes 100 µL of supernatant were transferred into the wells of a flat-bottom plate and optical density in the wells was measured at 405 nm. The activity of compounds was considered as their ability to suppress the destruction of membranes and thus decrease the concentration of free hemoglobin and optical density in the wells comparing to the control wells without additives. The activity of HA was calculated as (ODc-ODb)/(ODp-ODb)×100%, where ODp and ODs are mean optical densities in the wells with PBS and compound under investigation, correspondingly, and ODb (background) is mean optical density in the wells with erythrocytes, but without virus and compounds. The activity of HA in control wells without any virus was calculated by comparison to HA activity of influenza A virus.
4.3 Molecular docking study
The crystal structure of haemagglutinin was used for the docking procedure (PDB code: 3LZG) [23]. The geometric parameters of HA protein were optimised using the OPLS3 force field algorithm [24].
To find the active site in the HA corresponding to 3LZG PDB code, we analysed the binding site of the native ligand well-known in the HA inhibitor, TBHQ in 3EYM20 PDB code. This necessary procedure is one of the stages of molecular modelling. The TBHQ binding site is located at the interface between two monomers of the HA trimer. Based on an analysis of the active site, the area around the native ligand, with a radius of 5Å was selected. The functional amino acid sequence was compared in two PDB codes and the area of the TBHQ binding was detected in the protein 3LZG and was used for further docking procedures
The CPH-binding site was arranged at the site of proteolysis next to the amino acid valine at position 615 (the numbering corresponds to the 1RU7 [25] code). Details have been previously described [21].
Ligand preparation (optimisation, which took into account all possible conformations of the ligand) and all the docking procedures were carried out using the Schrodinger Small-Molecule Drug Discovery Suite 2018-4, Schrödinger, LLC, New York, NY, 2018 program packages. The docking was performed under the following conditions: ligand and protein are flexible and induced fit docking protocols were used for standard prediction accuracy.