Evaluation of antiviral activity of terpenophenols and some of their N- and O-derivatives

A comparative evaluation of the antiviral activity of a number of new and previously synthesized terpenophenols and their N- or O-containing derivatives against the A/Puerto Rico/8/34 (H1N1) virus strain was carried out. 2-Isobornylphenol, 1,2-dihydroxy-6-isobornyl-4-methylbenzene, 2-isobornyl-1,4-benzoquinone, and N-butyl-4-hydroxy-3,5-diisobornylbenzamide showed the highest activity.

The search for antiviral agents among natural compounds, which can be isolated from available and renewable plant materials, and their derivatives is a modern innovative approach to the development of new antiviral drugs. Plant-produced bioactive secondary metabolites, which include monoterpenoids and terpenophenols, are of interest as matrices for synthetic and semi-synthetic structural modifi cations, synthesis of combinatorial libraries of compounds, and study of the structure-activity relationship. Almost all compounds used as anti-fl u drugs, including Remantadine, Amantadine, Favipiravir, Oseltamivir (Tamifl u), Zanamivir (Relenza), and Ingavirin, are polyfunctional cyclic hydrocarbons having a small framework and branched side chains and containing NH 2 , NH, and OH groups. 1 It is known that compounds with a bornane (bicyclo-[2.2.1] heptane) structure, which include isobornylphenols, have antioxidant activity, as well as membraneprotective and cardioprotective, membrane stabilizing, hemorheological, and antithrombogenic properties. 2-10 Some representatives of isobornylphenols are used in medicine for the treatment of respiratory diseases 11 , showing antimicrobial 12 and antiviral activity. 13 The introduction of various substituents into the structures of organic molecules underlies the design of new drugs. For instance, the presence of the aminomethyl substituent in a molecule of phenolic compounds can lead to the appearance of antiviral activity. 14 For the synthesis of Mannich bases, 1-adamantylamine, which is an active substance for the prevention and treatment of the infl uenza A virus, can serve as an amine component. 1 There are examples of the synthesis of eff ective antiviral agents containing terpene and adamantyl substituents in their molecules. 15 This paper presents the results of a primary assessment of antiviral activity of a number of terpenophenols with isobornyl, bornyl, and isocamphyl substituents and their Nor O-containing derivatives and the analysis of the structure-property relationships for these compounds.

Results and Discussion
Compounds 1-38 were chosen as the objects of this study. Based on previously synthesized terpenophenols 1-6, derivatives 7-12 were obtained by hydroxymethyl ation using paraform aldehyde and boric acid (Scheme 1). Terpenophenols 1-6, 13-19 and their derivatives 29, 30 were synthesized according to known methods. 8, [16][17][18][19] Hydrochlorides 21-28 were prepared to increase the solubility of previously prepared Mannich bases 6,7,20 in DMSO. Compounds 13-30 are racemates. By condensation of substituted isobornylbenzaldehydes with 1-adamantylamine followed by reduction (in situ) of intermediate imines, aminomethyl derivatives containing the 1-adamantyl group at the nitrogen atom were synthesized for the fi rst time; they were also converted into the corresponding hydrochlorides 27 and 28. The synthesis of compound 27 is shown in Scheme 2 as an example.
The 1 H and 13 C NMR spectra, IR spectra, and the elemental analysis data obtained for new products 8, 9, 11, 12, 21-28, and 37 correspond to the expected structures. In the 1 H and 13 C NMR spectra of these compounds, the signals of protons and carbon atoms of the substituted terpenophenolic skeleton were observed. The SSCC value (J = 16.0 Hz) in the 1 H NMR spectrum of compound 37 makes it possible to identify this compound as having the E-confi guration of substituents relative to the double bond of the nitrovinyl fragment. The evaluation of antiviral activity of compounds 1-38 against the A/Puerto Rico/8/34 (H1N1) infl uenza strain in vitro was performed for the fi rst time (Table 1). In general, the studied compounds are characterized by high cytotoxicity except for compounds 22, 32, 34, 35, 38, for which CC 50 > 600 mol L -1 .
The lowest toxicity was exhibited by 2-(di methylaminomethyl)-6-isobornyl-4-methylphenol 22 with CC 50  880 mol L -1 . However, all non-toxic derivatives showed no signifi cant virus-inhibiting activity except benzamide 34 with two isobornyl substituents (selectivity index SI = 21). The low toxicity of derivatives with two isobornyl substituents in aromatic ring, with the exception of carboxyl derivatives 33 and 36, is consistent with previous results, 21 according to which the introduction of a second bulky substituent into an isobornylphenol molecule leads to a decrease in its toxicity.
Taking phenols 1-6 and their hydroxymethyl derivatives 7-12 as an example, it is shown that the structure of the terpene substituent in this chemical system does not substantially aff ect the virus-inhibiting activity. Hydroxymethyl derivatives with the isocamphyl substituent in the aromatic ring (compounds 9 and 12) showed the lowest cytotoxicity (CC 50 = 173 and 158 mol L -1 , respectively), at the same time, these compounds show no antiviral activity. 2-Isobornylphenol (1) and 2-isocamphylphenol (3) showed moderate antiviral activity (SI = 10 and 8, respectively). The presence of the methyl and/or hydroxyl group in  Note. CC 50 (M±SD) is the 50% cytotoxic concentration, which is a compound concentration reducing the optical density in the wells of cell culture plates by a factor of two compared to that of control wells, in which the compound was not added; IC 50 (M±SD) is a compound concentration resulting in the 50% reduction in the cytodestructive action of the virus; selectivity index (SI) is the ratio of CC 50 to IC 50 .
The data obtained indicate that within the studied set of derivatives, a substantial modifi cation of the skeleton of 2-isobornylphenol, including the introduction of nitrogen-containing substituents of diff erent complexity, does not result in an increase in the eff ect against the A/Puerto Rico/8/34 strain. The highest antiviral activity in this set of derivatives was found for compounds 27 and 28 containing the 1-adamantylamino group (SI = 5 and 6, respectively), which, however, exhibit rather high toxicity (9 and 3 mol L -1 ). When evaluating pairs of ortho/para isomers 22 and 23, 25 and 26, 27 and 28, it was found that the presence of the aminomethyl group in the para position led to an increase in the cytotoxicity of the compounds.
The antiviral potential of the most active compounds (20 and 34) and phenol 1, which is the least substituted compound, was additionally assessed against other human respiratory viruses: coronavirus OC43, human parainfl uenza virus type 3, and human adenovirus type 5 ( Table 2). As can be seen, the virus-inhibiting activity of these compounds against human respiratory viruses was lower than against human infl uenza viruses. At the same time, enveloped viruses (coronavirus and parainfl uenza virus) turned out to be more sensitive to the action of isobornylphenol derivatives than non-enveloped adenovirus, however, none of the studied compounds blocked their reproduction to a level of SI > 10.
Despite the fact that the stud ied compounds showed rather moderate antiviral activity, we were able to identify the leading compounds among them. Note that the cytotoxicity of phenolic compounds represents a substantial barrier to the development of new candidates for drug. The data obtained contribute to the basis for the design of new compounds with improved cytotoxicity and preserved antiviral properties.

Experimental
The analysis of the synthesized compounds was partly performed using the equipment of the Center of Collective Usage "Chemistry" of the Institute of Chemistry, Komi Scientifi c Center, Ural Branch of the Russian Academy of Sciences. 1 Н and 13 С NMR spectra of the prepared compounds were recorded on a Bruker Avance II 300 spectrometer (300.17 and 75.5 MHz) in DMSO-d 6 and CDCl 3 . The assignment of 1 H signals was carried out using NOESY, that of 13 C signals was performed on the basis of 13 С NMR spectra recorded in the J-modulation mode, as well as on the basis of HSQC and HMBC experiments. Diff use refl ectance IR spectra were recorded on a Shimadzu IR Prestige 21 FTIR spectrometer in KBr pellets (solid compounds) or in thin layers (liquid compounds). Elemental analysis was carried out on a vario Micro cube analyzer. Melting points were determined using a Sanyo Gallenkamp MPD350 device, no corrections were applied. An Optical Activity polAAr 3001 polarimeter was used to measure the specifi c rotations. The progress of the reactions was monitored by TLC on Sorbfi l plates (JSC IMID, RF). The chromatographic zones of the reaction products were detected by treating the plates with a solution of KMnO 4 (15 g of KMnO 4 , 300 mL of H 2 O, and 0.5 mL of concentrated H 2 SO 4 ). Preparative chromatographic separation was carried out on columns fi lled with silica gel (0.06-0.2 mm, Alfa Aesar).

2-(1,7,7-Trimethylbicyclo[2.2.1]hept-exo-2-yl)cyclohexa-2,5-dien-1,4-dione (20).
Synthesis of compounds 21-26 (general procedure). To a solution of the corresponding amine (1.0 mmol) in Et 2 O (5-20 mL), a 2 M HCl solution in EtOH was added dropwise until pH <6, and the mixture was vigorously stirred at room temperature for 30-60 min. The precipitated hydrochloride was fi ltered off (with the exception of compound 24), washed with a little amount of chilled Et 2 O and dried. To isolate dihydrochloride 24, the ether solution was separated, the oily residue was successively washed with chilled Et 2 O (6 mL) and pentane (2×6 mL); the solvents were removed under reduced pressure; the residue was dried. Cells were seeded into 96-well cell culture plates (Biologix, China, cat. No. 07-6096) in an amount of 10 4 cells per well and cultivated until the formation of a confl uent monolayer. The resulting cultures were subsequently used to assess the antiviral and cytotoxic activity of the compounds.
Evaluation of the cytotoxic eff ect of the test comounds in cell culture (the MTT assay). The study of the toxicity of the compounds was carried out on the basis of the assessment of the viability of the cells of the corresponding lines using the reduction reaction of the tetrazolium dye MTT by cells in culture, the intensity of which refl ects the degree of cell viability as a result of the reduction of the dye by mitochondrial and partially cytoplasmic dehydrogenases.
The test compounds at concentrations of 3.7-300 g mL -1 dissolved in the cell cultivation medium were introduced into wells of a cell culture plate in the amount of 200 L, and the plates were incubated for 72 h at 36 С in an atmosphere of 5% СО 2 . Then the cells were washed with the MEM medium, and 100 L of a solution (0.5 mg mL -1 ) of the tetrazolium dye MTT in the cell culture medium was added into each well. The cells were incubated at 36 С in an atmosphere of 5% СО 2 for 2 h and, then, washed with physiologic saline for 5 min. The residue in each well was dissolved in 100 L of DMSO, and optical density was measured using a Thermo Scientifi c Multiscan FC microplate photometer at  = 540 nm. Based on the data obtained, the 50% cytotoxic concentration (СС 50 ) was calculated, which is the compound concentration reducing the optical density in the well by a factor of two as compared to the control wells, into which the compound was not added.
Study of antiviral activity of compounds. The test samples (100 L) were added to the wells of cell culture plates containing monolayers of cells of the respective line. The cell culture plates with cells were incubated in an atm osphere of 5% CO 2 at 37 С for 1 h. After that, 0.1 mL of the virus in α-МЕМ medium was introduced into the wells at a multiplic-ity of infection of 0.01 TCID 50 per cell, and the cell culture plates with cells were incubated in an atmosphere of 5% CO 2 at 36 С for 72 h. The infected cells were then washed with MEM medium and cell viability assay was performed as described above. Based on the data obtained, the 50% inhibitory concentration (IC 50 ) resulting in a 50% reduction in viral cell destruction was calculated for each compound.
Calculations of 50% cytotoxic (CC 50 ) and 50% in hibitory (IC 50 ) concentrations were performed using the GraphPad Prism 6.01 software package. A 4-parameter equation of the logistic curve was taken as a working model for analysis (menu options "Nonlinear regression"-"inhibitor logarithm-response"). Based on the data obtained, for each compound and each virus, the selectivity index (SI), which is the ratio of CC 50 to IC 50 , was calculated.