Stereoselective Synthesis of Novel Adamantane Derivatives with High Activity Against Rimantadine-Resistant Influenza A Virus Strains

Stereoselective Synthesis of Novel Adamantane Derivatives with High Activity Against Rimantadine-Resistant Influenza A Virus Strains Nikolai Yu. Kuznetsov,*[a] Rabdan M. Tikhov,[a] Ivan A. Godovikov,[a]† Michail G. Medvedev,[a]‡1 Konstantin A. Lyssenko,[a] ‡ Elena I. Burtseva,[b] Elena S. Kirillova,[b] and Yuri N. Bubnov[a][c] [a] A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilov 28, 119991, Moscow, Russian Federation [b] N. F. Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medicinal Sciences, Gamaleya 18, 123098, Moscow, Russian Federation [c] N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky pr. 47, 119991 Moscow, Russia * nkuznff@ineos.ac.ru Supporting Information

Instead of purification of the residue after evaporation of the reaction mixture, its solution in EtOAc was rapidly passed through the 0.5 cm layer of silica gel on the filter funnel connected to a vacuum line and washed in EtOAc.EtOAc was evaporated, residual oil dissolved in n-C 6 H 14 and again evaporated to remove traces of EtOAc.The resulting transparent oil consisting of isomeric bromides 9b (75%) was diluted with n-C 6 H 14 and placed in refrigerator at -25 °С for crystallization.The precipitated crystals were filtered and washed with chilled n-C 6 H 14 , that gave pure cis-9b (8%), m.p. 143-144 °С. 1   To a solution of crude mixture of the isomeric bromides 9b (0.52 g, 1.35 mmol) in THF (15 mL) at 0 °С was added tBuOK (0.19 g, 1.70 mmol).The mixture was then stirred for 1 h at ambient temperature.

Antiviral activity assay
We used influenza A viruses: etalon pandemic strain A/California/7/2009 (H1N1)pdm09 and modern pandemic strain A/IIV-Orenburg/29-L/2016 (H1N1)pdm09 that were resistant to rimantadine.The latter strain induced severe form of Influenza A infection with a lethal outcome.
Cell immuno-enzymatic analysis (IEA) MDCK cells monolayer in 96-well plates were treated with adamantane derivative at 6 different concentrations in 6 wells of repeat.After then dilutions of virus were added to the corresponding wells in working doses 100-1000 TCID 50 /ml.After 24 h incubation at 37 °C under 5% CO 2 atmosphere the cells were fixed with 80% acetone in phosphate buffer.Cell immuno-enzyme analysis (IEA) was carried out according to the literature [1].The percentage of virus activity inhibition by the test compounds was evaluated by the formula: where OD is optical density at 450 nm, OD cc is OD 450 of cell control, and OD vc is OD 450 of virus control.Compound 10 (10.8 mg) were dissolved in THF-D 8 (1.2 ml).This solution (0.3 ml) was mixed in a vial with MEM medium (0.3 ml) and additional volume of THF-D 8 (0.15 ml) was added to prevent the separation of 10 from the solution.After then it was transferred to NMR tube for registration of spectra.
The sample was heated on the water bath at 37 C and the multiple proton spectra with water peak suppression were recorded in 1.5-2.0h intervals until the conversion of 10 reaches 50%.Integration of the intensity of signal of olefin 10 was performed at 2.92 ppm (triplet) respectively to signals of a component of MEM medium at 2.81 ppm and additionally to residual THF peak at 1.77 ppm.
From the results was built a graph having trend line y = -0.0677x+ 1.0035 (R 2 = 0.9989) from which calculated T 1/2 for 10 = 7.44 hours.

X-ray structures determination.
The X-ray crystal structure analyses were carried out on Bruker SMART APEX2 CCD (10-12; MoK  radiation, graphite monochromator,  and  scan mode) and on Bruker SMART APEX2 DUO CCD ((S)-8; CuK α radiation,  and  scan mode).The data sets were corrected for absorption with SADABS [1] and crystal structures were solved with the ShelXT [2] structure solution program using Direct Methods and refined with the ShelXL [3] refinement package using Least Squares minimization.Nonhydrogen atoms were refined anisotropically.The absolute structure for (S)-8 were defined by the refinement of the Flack parameters.The hydrogen atoms of the amino groups were localized in the difference-Fourier maps and refined isotropically with displacement parameters [U iso (H) = 1.2U eq (N)].
The other hydrogen atoms were placed in calculated positions and refined within riding model with fixed isotropic displacement parameters [U iso (H) = 1.2U eq (C)].
In order to obtain electron density function we have used the Hansen-Coppens formalism [4] as implemented in the program package XD.[5] Input files were generated with the program InvariomTool.
[6] Topological analysis of the invariom electron density distributions obtained from X-ray diffraction data in the harmonic approximation was carried out using the WINXPRO program package.[7] Potential energy density v(r) was evaluated through the Kirzhnits's approximation [8] for kinetic energy density function g(r).Accordingly, the g(r) function is described as , giving in conjunction with the virial theorem (2g(r)+ν(r)=1/4 2 (r)) [9] the expression for v(r).Interaction energies were estimated by means of the Espinosa's correlation scheme -a semiquantitative relation between the energy of an interaction and the value of the potential energy density function v(r) in its bcp.[10] Having a very simple form as 0.5v(r), it was repeatedly shown to give accurate estimates in many cases (those are succinctly summarized in [11]).

Fig. S3 .
Fig. S3.The general view of N-H…O and C-H…O bonded chains in the crystal of (R)-11 in representation of atoms by thermal ellipsoids (p=50%).The presence of all interactions shown was proved by the topological analysis of electron density obtained by means of invariom model.

Fig. S4 .
Fig. S4.The general view of N-H…O bonded chains in the crystal of (4R,6R)-12 in representation of atoms by thermal ellipsoids (p=50%).The anisotropy of atomic displacement parameters of OH group excludes the possibility to analyze the interatomic interactions by means of invariom model.