Bicyclic monoterpenoids are a source of organic compounds with a unique structure that provides them with various biological effects. In particular, the gem-dimethyl moiety, which is present in all bicyclic monoterpenoids, promotes the formation of van der Waals interactions with the protein binding site [1]. Therefore, the synthesis of compounds incorporating a natural fragment serves as an effective strategy in drug development. Previously, our research group paid considerable attention to the synthesis and study of the antiviral properties of nitrogen-containing derivatives of bicyclic monoterpenoids. A study of (–)-borneol esters having the general structure I (Fig. 1) revealed derivatives with a wide spectrum of antiviral activity. Substances have been discovered that inhibit influenza A virus [2], Ebola virus (Zaire strain), and Marburg (Popp strain) [3], respiratory syncytial virus [4], and SARS-CoV-2 virus [5]. Ethers II (Fig. 1), synthesized from camphene by an acid-catalyzed Wagner-Meerveen rearrangement in the presence of K10 clay, also demonstrated significant virus inhibitory effects against influenza A (H1N1) virus, Ebola virus, and Hantaan virus [6]. Also, based on (1S)-(+)-camphor-10-sulfonic acid, a number of new sulfonamides III containing various substituents at the nitrogen atom were synthesized and their antiviral properties against the Ebola and Marburg viruses were studied [7, 8].

Fig. 1.
figure 1

General structures of previously studied bicyclic monoterpenoids with antiviral activity. H1N1—influenza virus, EBOV—Ebola virus, RSV—respiratory syncytial virus, MARV—Marburg virus.

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In continuation of these studies on the synthesis of potential antiviral agents, this work examines the possibility of synthesizing compounds based on (1S)-(+)-camphor-10-sulfonic acid, (+)-ketopinic and (–)-camphanic acids, having the structural type schematically shown in Fig. 2. The listed derivatives retain in their structure the gem-dimethyl group and the bicyclic structure of the natural skeleton. The ethyl fragment was chosen as a linker, since, according to previous results, compounds with two methylene units are often characterized by lower toxicity. Morpholine, piperidine, and N-substituted piperazines were chosen as the nitrogen-containing component.

Fig. 2.
figure 2

Strategy for the synthesis of monoterpenoid derivatives studied in this work.

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(1S)-(+)-Camphor-10-sulfonic acid 1 does not have high chemical reactivity, including due to low solubility in organic solvents. Therefore, in the first stage, sulfonic acid 1 was converted into sulfonyl chloride 2. For the subsequent introduction of the heterocyclic fragment, interaction with 2-bromoethanol was carried out to obtain sulfonate 3. At the final stage, it was planned to carry out nucleophilic substitution of the bromine atom with secondary amines. Thus, sulfonate 3 was reacted with morpholine in the presence of a base. As the reaction result, instead of the expected product 4, product 5 and the starting bromide 3 were found (Scheme 1). The formation of product 5 can be explained by nucleophilic substitution at the carbon atom in the ethyl linker, where camphor-10-sulfonic acid 1 plays the role of a good leaving group. Thus, it was not possible to replace the bromine atom in compound 3 with morpholine, since the sulfo group turned out to be extremely prone to elimination under the influence of an N-nucleophile.

Scheme
scheme 1

1.

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Another direction of modification of camphor-10-sulfochloride 2 is oxidation to ketopinic acid 6. For oxidation, we chose a method using potassium permanganate and sodium carbonate [9], following which acid 6 was obtained in 65% yield. Next, the resulting carboxylic acid was converted into acid chloride 7, which was isolated in its individual form and used in further reactions without additional purification. In the next stage, the reaction of acid chloride 7 with bromoethanol led to product 8, which was isolated and purified by column chromatography. At the final stage, the reaction of bromide 8 with morpholine, piperidine and 4-methylpiperazine led to the corresponding esters 9a–9d, including a nitrogen-containing heterocyclic fragment (Scheme 2). This transformation occurs with complete conversion and the yield of crude reaction products is 85–90%. NMR and CMS spectra of unpurified products 9a–9d confirm the structure of the obtained substances. However, when performing column chromatography on silica gel (60–200 mesh, Masherey-Nagel), using a mixture of hexane–ethyl acetate–methanol (1%) as an eluent, partial destruction of esters 9a–9d with the formation of methyl ester of ketopinic acid is observed. The yield of target products after column chromatography is no more than 15%. Apparently, a side process of transesterification occurs under the influence of methanol, which is used as one of the components of the eluent. It is known that silica gel has a slightly acidic character, which facilitates the transesterification process.

Scheme
scheme 2

2.

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A similar strategy for the construction of nitrogen-containing heterocyclic derivatives was used for the synthesis of (–)-camphanic acid esters (Scheme 3). Camphanic acid is actively used as a chiral auxiliary for the separation of racemic mixtures, and its synthesis is described in sufficient detail in a number of publications starting from camphoric acid [10]. In this work, commercial (–)-camphanic acid was used. At the first stage, acid chloride 11 was obtained, which at the next stage was converted into ester 12, containing a bromine atom capable of nucleophilic substitution. At the final stage, the reaction of ester 12 with morpholine, piperidine, and N-phenylpiperazine led to the corresponding products 13a–13c.

Scheme
scheme 3

3.

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Esters 13a–13c, by analogy with ketopinic acid esters 9a–c, have been to be unstable under the conditions of column chromatography on silica gel. Thus, after column chromatography of compound 13a, (–)-camphanic methyl ester 14 and alcohol 15 were isolated (Scheme 4). The yield of the target product 13a relative to the initial mass of the crude product was 11%.

Scheme
scheme 4

4.

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Thus, the use of a synthesis strategy involving the preparation of a bromo-substituted ester followed by substitution with a nitrogen-containing heterocyclic moiety leads to the target esters of (+)-ketopinic and (–)-camphanic acids. However, the synthesized products turned out to be unstable under column chromatography conditions due to the side transesterification process.

Quantum chemical calculations of the reactivity of compounds 9a–9d, 13a–13c, and (–)-borneol esters. Analysis of experimental data on the stability of bicyclic monoterpenoids derivatives showed that (–)-borneol esters with structure I (Fig. 1) are stable under conditions of column chromatography on silica gel (60–200 mesh, Masherey-Nagel) using a mixture as an eluent hexane–ethyl acetate–methanol. At the same time, esters of (+)-ketopinic acid 9a–9d and (–)-camphanic acid 13a–13c undergo transesterification under the influence of methanol, which is used as an eluent component, to form the corresponding methyl esters. To identify possible factors determining the stability of esters of bicyclic monoterpenoid derivatives, quantum chemical calculations were carried out. Three compounds from each group of derivatives Ia, 9a, and 13a were selected as the subject of the study (Scheme 5).

Scheme
scheme 5

5.

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The mechanism of the transesterification reaction can be considered by analogy with the acid-catalyzed hydrolysis of esters, which includes 4 types: ААС1, ААL1, AAC2, and AAL2. In the AAC1 mechanism, the ester is protonated at the alkyl oxygen, resulting in cleavage of the acyl-oxygen bond and the formation of acylium ions (intermediate A, Scheme 6). It is believed that this mechanism is rare and characteristic of a strongly acidic environment. However, the AAC1 mechanism is typical for compounds where the R1 group is very large, which prevents bimolecular attack [11]. Since in the case of compounds 9a and 13a, the R1 group is sterically bulky, we included the AAC1 mechanism as a possible transesterification pathway. Traditionally, the transesterification of esters in an acidic environment should proceed through the AAC2 mechanism. In this case, after protonation of the carbonyl group, a nucleophilic attack by an alcohol molecule occurs, leading to tetrahedral intermediate B and its subsequent decomposition. The stages set of a possible transesterification mechanism is shown in Scheme 6 [12].

Scheme
scheme 6

6.

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To analyze the reactivity of the studied esters, we calculated the thermodynamic parameters: the standard enthalpy of the reaction (ΔrH°) and the standard Gibbs free energy of the reaction (ΔrG°) for the stage of carbocations A and B formation, i.e., the limiting stages of the mechanisms presented in Scheme 6. To assess the internal strength of the C–O bond, we used the IBSI (Intrinsic Bond Strength Index) [13], calculated in the MultiWFN program [14].

The thermodynamic parameters of the reaction for the carbocations A and B formation for compounds 13a and 9a are greater than for compound Ia. The resulting series of changes in thermodynamic parameters corresponds to the observed experimental data, according to which (–)-borneol esters are more stable in comparison with (+)-ketopinic acid and (–)-camphanic acid esters. The values of the IBSI parameter also increase in the series 13a, 9a, and Ia, which indicates that the C–O bond in compound Ia is stronger compared to that in compounds 9a and 13a.

Thus, the greater stability of (–)-borneol esters may be due to a stronger C–O bond and, in addition, the calculated thermodynamic parameters also indicate a lower tendency of (–)-borneol esters to transesterification under column chromatography conditions in comparison with esters of (+)-ketopinic acid 9a and (–)-camphanic acid 13a.

Assessment of antiviral activity in vitro against influenza virus. The cytotoxicity of (+)-ketopinic acid esters 9a–9d and (–)-camphanic acid esters 13a–13c and their ability to inhibit the reproduction of influenza virus A/Puerto Rico/8/34 in vitro was determined. The drug ribavirin was used as a positive control in the study. Cytotoxic properties were determined against the MDCK cell line using the standard MTT method.

As shown in Table 1, among the studied derivatives, only the (+)-ketopinic acid ester 9b, which includes a piperidine ring, showed a moderate virus inhibitory effect and low cytotoxicity on MDCK cell culture. Compound 9d also showed moderate antiviral activity, while showing higher cytotoxicity. Among camphanic acid derivatives 13a–13c, ester 13a with a morpholine fragment turned out to be non-toxic and inactive, esters 13b, c showed a noticeably more pronounced toxic effect and did not demonstrate antiviral activity.

Table 1. Antiviral activity of compounds 9a–9d and 13a–13c against influenza virus A/Puerto Rico/8/34a
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Thus, esters of (+)-ketopinic and (–)-camphanic acids were synthesized, including the cycle of morpholine, piperidine, N-methylpiperazine, and N-phenylpiperazine. It has been shown that these esters are unstable under column chromatography conditions. A study of antiviral properties revealed that the ester of (+)-ketopinic acid with a piperidine fragment 9b exhibits a moderate inhibitory effect against the influenza virus A/Puerto Rico/8/34. It has been indicated that (+)-camphor-10-sulfonic acid bromoethyl ester reacts with N-centered nucleophiles, forming a disubstitution product with the elimination of camphor-10-sulfonic acid.

EXPERIMENTAL

1H and 13C NMR spectra were recorded on Bruker AV-300 spectrometer [operating frequencies 300.13 (1H), 75.47 MHz (13C)], AV-400 [400.13 (1H) and 100.78 MHz (13C)]. Chemical shifts are given relative to the CDCl3 solvent signal. High-resolution mass spectra were recorded on DFS ThermoScientific and Agilent 7200 Accurate Mass Q-TOF spectrometers in full scanning mode in the range m/z 0–500, electron impact ionization 70 eV with direct sample injection. The reaction progress monitoring and the purity of the compounds was carried out by chromatography-mass spectrometry on an Agilent 7890 A gas chromatograph with an Agilent 5975C quadrupole mass spectrometer as a detector, HP-5MS quartz column 30000×0.25 mm, carrier gas – helium. Elemental analysis was performed on a Euro EA 3000 C,H,N,S analyzer. A Mettler Toledo FP900 thermal system was used to measure the melting point. Separation and isolation of reaction products were carried out using column chromatography on silica gel (60–200 mesh, Masherey-Nagel).

{(1S,4R)-7,7-Dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl}methanesulfonyl chloride (2). The mixture of (1S)-(+)-camphor-10-sulfonic acid 1 (5 g, 21 mmol) and thionyl chloride (10 mL, 74 mmol) was refluxed for 3 h. Next, excess thionyl chloride was removed by distillation. Since sulfonyl chloride 2 is a fairly thermolabile substance, the heating of the reaction mixture must be carefully controlled. Yield 93%, light brown solid substance, mp 65°С. 1H NMR spectrum (CDCl3), δ, ppm: 0.90 s (3H, 8-Me), 1.11 s (3H, 9-Me), 1.42–1.49 m (1H, H5endo), 1.71–1.79 m (1H, H6endo), 1.96 d (1H, H3endo, J 18.7 Hz), 2.02–2.11 m (1H, H5exo), 2.13 br. t (1H, H4, J 4.9 Hz), 2.37–2.47 m (2H, H3exo, H6exo), 3.70 d (1H, H10, J 14.5 Hz), 4.28 d (1H, H10′, J 14.5 Hz).

2-Bromoethyl-{(1S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl}methanesulfonate (3). 2-Bromoethanol (0.21 mL, 3 mmol) and K2CO3 (7 mmol) were added to a solution of (1S)-(+)-camphor-10-sulfonyl chloride 2 (0.7 g, 2.8 mmol) in CH2Cl2 (5 mL). The reaction mixture was stirred at low heat (75°C) for 12 h, and then a saturated NaCl solution was added and extracted twice with CH2Cl2. The combined organic layer was dried with anhydrous Na2SO4, and the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography on silica gel [eluent: hexane–ethyl acetate (5–15%)–methanol (2%)]. Yield 53%, pale brown solid substance, mp 54.2–57.4°С. 1H NMR spectrum (CDCl3), δ, ppm: 0.85 s (3H, 8-Me), 1.08 s (3H, 9-Me), 1.38–1.46 m (1H, H5endo), 1.64–1.72 m (1H, H6endo), 1.94 d (1H, H3endo, J 17.9 Hz), 1.98–2.08 m (1H, H5exo), 2.1 br. t (1H, H4, J 4.5 Hz), 2.31–2.48 m (2H, H3exo, H6exo), 3.03 d (1H, H10, J 13.2 Hz), 3.52–3.57 m (2H, Н12), 3.60 d (1H, H10′, J 13.2 Hz), 4.45–4.58 m (2Н, Н11). 13С NMR spectrum (CDCl3), δС, ppm: 214.2 (C2), 68.4 (C11), 57.7 (C1), 47.2 (C10), 42.8 (C7), 42.6 (С4), 42.3 (C3), 27.6 (C12), 26.6 (С6), 24.7 (C5), 19.5 (8-Мe, 9-Мe). Mass spectrum (HRMS), m/z: 338.0175 [M]+ (calculated for C12H19O4Br1S1: 338.0182 [M]+).

Synthesis of (+)-ketopinic acid (6). A solution of (1S)-(+)-camphor-10-sulfonic acid chloride (20.0 g, 80.0 mmol) in MeCN (50 mL) was added to a suspension consisting of Na2CO3 (24.8 g), KMnO4 (27.1 g, 171.5 mmol), H2O (200 mL), and MeCN (100 mL). The resulting mixture was stirred for 30 min at room temperature, and then stirring was continued for 2 h at 70°C. After cooling the mixture to room temperature, a mixture of 3 M H2SO4 (136.5 mL) and 2 M Na2SO3 (320 mL) was added dropwise to the solution to avoid excessive foaming. Additional 3 M H2SO4 was added until the solution became clear. Extraction was performed with Et2O (3×100 mL); the combined organic layers were washed with brine and dried with Na2SO4. The solvent was removed in vacuum. Yield 71%, white solid substance. 1H NMR spectrum (CDCl3), δ, ppm: 1.10 s and 1.16 s (2×3H, 8-Me and 9-Me), 1.42 d. d. d (1H, H5endo, 2J 12.5, J5endo,6endo 9.1, J5endo,6exo 3.7 Hz), 1.73–1.82 m (1H, H6endo), 1.99 d (1H, H3endo, J 19.6 Hz), 2.03–2.09 m (1H, H5exo), 2.1–2.14 m (1H, H4), 2.33–2.42 m (1H, H6exo), 2.56 d. t (1H, H3exo, 2J 18.3, J3exo,4 4.0 Hz). 13С NMR spectrum (CDCl3), δС, ppm: 213.9 (C2), 173.7 (C10), 66.4 (C1), 49.7 (C3), 43.7 (C4), 43.3 (C7), 27.1 and 26.6 (C6, C5), 20.5 and 19.7 (C9 and C8).

Ketopinic acid chloride (7) was prepared according to a previously described procedure [9]. Yield 75%, solid yellow substance. 1H NMR spectrum (CDCl3), δ, ppm: 1.13 s and 1.16 s (2×3H, 8-Me and 9-Me), 1.42–1.49 m (1H, H5endo), 1.95–2.15 m (4H, H6endo, H3endo, H5exo, H4), 2.41–2.51 m (1H, H6exo), 2.51–2.60 m (1H, H3exo).

(1S,4R)-2-Bromoethyl-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-carboxylate (8). 2-Bromoethanol (0.3 mL, 4 mmol) and K2CO3 (0.8 g, 6 mmol) were added to a solution of (1S)-(+)-ketopinic acid chloride 7 (0.6 g, 3 mmol) in CH2Cl2 (5 mL). The reaction mixture was stirred at room temperature for 12 h. After the reaction completion, NaCl solution was added to the mixture and extracted with CH2Cl2 (3×10 mL). The combined organic layer was dried with Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent: hexane–ethyl acetate). Yield 45%, yellow oily substance. 1H NMR spectrum (CDCl3), δ, ppm: 1.07 s and 1.15 s (2×3H, 8-Me and 9-Me), 1.38 d. d. d (1H, H5endo, 2J 12.9, J5endo,6endo 8.8, J5endo,6exo 4.1 Hz), 1.69–1.83 m (1H, H6endo), 1.90 d (1H, H3endo, J 18.3 Hz), 1.96–2.05 m (1H, H5exo), 2.08 br. t (1H, H4, J 4.4 Hz), 2.30–2.40 m (1H, H6exo), 2.51 d. t (1H, H3exo, 2J 18.1, J3exo,4 3.8 Hz), 3.49 t (2H, H12, J 5.7 Hz), 4.36–4.50 m (2H, Н11). 13С NMR spectrum (CDCl3), δС, ppm: 210.5 (C2), 169.2 (C10), 67.8 (C1), 63.8 (C11), 49.2 (C3), 44.2 (C4), 43.6 (C7), 28.6 (C12), 26.1 (C6, C5), 21.1 and 19.5 (C9 and C8). Mass spectrum (HRMS), m/z: 288.0351 [M]+ (calculated for C12H17O3Br1: 288.0356 [M]+).

General procedure for synthesis of compounds 9a–9d. The corresponding amine (1.3 eq.) and K2CO3 (3 eq.) were added to a solution of bromide 8 (1 eq.) in MeCN (5 mL). The reaction mixture was stirred at room temperature for 12 h, then the solvent was evaporated under reduced pressure, the resulting residue was re-dissolved in CH2Cl2, washed with NaCl solution, and extracted twice with CH2Cl2. The combined organic layer was dried with anhydrous Na2SO4 and evaporated. The crude products were purified by chromatography on a silica gel column (eluent: hexane–ethyl acetate).

(1S,4R)-2-Morpholinoethyl-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-carboxylate (9a). Yield 7%, yellow oily substance. 1H NMR spectrum (DMSO-d6), δ, ppm: 0.99 s and 1.05 s (2×3H, 8-Me and 9-Me), 1.30–1.41 m (3H, H5endo, 2J 12.5, J5endo,6endo 9.2, J5endo,6exo 3.5 Hz), 1 1.53–1.65 m (1H, H6endo), 1.83–1.97 m (1H, H5exo), 1.92 d (1H, H3endo, J 18.6 Hz), 2.05 br. t (1H, H4, J 4.4 Hz), 2.17–2.29 m (1H, H6exo), 2.32–2.39 m (4H, H13, H13′), 2.41–2.53 m (3H, H3exo, H12), 3.50 t (2H, H14, H14′, J 4.7 Hz), 4.04–4.28 m (2H, H11). 13С NMR spectrum (DMSO-d6), δС, ppm: 210.5 (C2), 169.2 (C10), 67.6 (C1), 66.1 (C14), 61.1 (C11), 56.5 (C12), 53.2 (C13, C13′), 48.9 (C3), 43.7 (C4), 43.3 (C7), 25.9 and 25.6 (C5, C6), 21.0 and 19.4 (C9 and C8). Mass spectrum (HRMS), m/z: 295.1782 [M]+ (calculated for C16H25O4N1: 295.1778 [M]+).

(1S,4R)2-(Piperidin-1-yl)ethyl-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-carboxylate (9b). Yield 15%, pale yellow oily substance. 1H NMR spectrum (CDCl3), δ, ppm: 1.05 s and 1.13 s (2×3H, 8-Me and 9-Me), 1.32–1.43 m (3H, H5endo, H15), 1.48–1.57 m (4H, H14, H14′), 1.69–1.80 m (1H, H6endo), 1.91 d (1H, H3endo, J 18.6 Hz), 1.94–2.05 m (1H, H5exo), 2.07 br. t (1H, H4, J 4.6 Hz), 2.28–2.50 m (6H, H6exo, H3exo, H13, H13′), 2.60 t (2H, H12, J 5.1 Hz), 4.17–4.35 m (2H, H11). 13С NMR spectrum (CDCl3), δС, ppm: 210.9 (C2), 169.5 (C10), 67.7 (C1), 61.9 (C11), 57.1 (C12), 54.5 (C13, C13′), 49.2 (C3), 44.2 (C4), 43.7 (C7), 26.2 and 26.1 (C5, C6), 25.7 (C14, C14′), 23.9 (C15), 21.1 and 19.5 (C9 and C8). Mass spectrum (HRMS), m/z: 293.1989 [M]+ (calculated for C17H27O3N1: 293.1986 [M]+).

(1S,4R)2-(4-Methylpiperazin-1-yl)ethyl-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-carboxylate (9c). Yield 10%, yellow oily substance. 1H NMR spectrum (CDCl3), δ, ppm: 1.02 s and 1.09 s (2×3H, 8-Me and 9-Me), 1.32–1.40 m (1H, H5endo), 1.67–1.76 m (1H, H6endo), 1.90 d (1H, H3endo, J 18.7 Hz), 1.98–2.03 m (1H, H5exo), 2.06 br. t (1H, H4, J 5.5 Hz), 2.24–2.34 m (1H, H6exo), 2.46–2.55 m (4H, H3exo, 15-Ме), 2.66 br. t (2H, H12, J 5.5 Hz), 2.69–2.97 br. m (8H, H13, H13′, H14, H14′), 4.16–4.32 m (2H, H11). 13С NMR spectrum (CDCl3), δС, ppm: 210.8 (C2), 169.4 (C10), 67.7 (C1), 61.3 (C11), 55.6 (C12), 53.0 and 50.3 (C13, C13′, C14, C14′), 49.1 (C3), 44.1 (C4), 43.6 (C7), 43.3 (C15), 26.1 (C5, C6), 21.1 and 19.5 (C9 and C8). Mass spectrum (HRMS), m/z: 308.2097 [M]+ (calculated for C17H28O3N2: 308.2094 [M]+).

(1S,4R)2-(4-Ethylpiperazin-1-yl)ethyl-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-carboxylate (9d). Yield 12%, pale yellow oily substance. 1H NMR spectrum (CDCl3), δ, ppm: 1.03 t (3H, 16-Ме, J 7.4 Hz), 1.05 s and 1.12 s (2×3H, 8-Me and 9-Me), 1.32–1.40 m (1H, H5endo), 1.69–1.78 m (1H, H6endo), 1.90 d (1H, H3endo, J 18.3 Hz), 1.94–2.05 m (1H, H5exo), 2.07 br. t (1H, H4, J 4.8 Hz), 2.27–2.57 m (12H, H6exo, H3exo, H15, H13, H13′, H14, H14′), 2.60 t (2H, H12, J 6.0 Hz), 4.18–4.36 m (2H, H11). 13С NMR spectrum (CDCl3), δС, ppm: 210.9 (C2), 169.5 (C10), 67.8 (C1), 61.8 (C11), 56.5 (C12), 53.1 and 52.6 (C13, C13′, C14, C14′), 52.1 (C15), 49.2 (C3), 44.2 (C4), 43.8 (C7), 26.2 and 26.2 (C5, C6), 21.2 and 19.6 (C9 and C8), 11.81 (C16). Mass spectrum (HRMS), m/z: 320.2096 [M – 2H]+ (calculated for C18H28O3N2: 320.2094 [M – 2Н]+).

(1S,4R)-4,7,7-Trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carbonyl chloride (11). To 0.5 g (2.5 mmol) of (1S)-(–)-camphanic acid 10, 1.8 mL (25.2 mmol) of thionyl chloride was added and the reaction mixture was refluxed for 3 h. Excess thionyl chloride was removed by distillation under reduced pressure. Yield 81%, pale yellow crystalline substance. The spectral characteristics are consistent with those presented in the literature [10].

2-Bromoethyl-(1S,4R)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate (12). To a solution of 1.80 g (8.3 mmol) of acid chloride 11 in 15 mL of CH2Cl2, 1.18 mL (16.6 mmol) of 2-bromoethan-1-ol and excess K2CO3 were added with stirring. The mixture was stirred at ~40°C for 12 h, and then a saturated NaCl solution was added and extracted with CHCl3. The combined organic layer was dried with anhydrous Na2SO4, the drying agent was filtered off, and the solvent was distilled off. The resulting product was purified by column chromatography on SiO2 using a mixture of hexane–ethyl acetate–methanol (1%) as an eluent. Yield 89%, colorless crystalline substance, mp 127.3°C. 1H NMR spectrum (CDCl3), δ, ppm: 0.96 s (3H, 10-Мe), 1.06 s (3H, 9-Мe), 1.09 s (3H, 8-Мe), 1.62–1.70 m (1H, H6endo), 1.87–1.94 m (1H, H6endo), 2.02 septet (1H, H6exo, J 4.6 Hz), 2.38–2.45 m (1H, H5exo), 3.54 t (2H, H13, J 5.9 Hz), 4.48–4.56 m (2H, H12). 13С NMR spectrum (CDCl3), δС, ppm: 177.6 (C3), 166.8 (C11), 90.8 (C7), 64.5 (C12), 54.6 (С1), 54.1 (C4), 30.5 (С6), 28.7 (C5), 27.9 (C13), 16.6 (8-Мe), 16.5 (9-Мe), 9.5 (10-Мe). Mass spectrum (HRMS), m/z: 304.0310 [M]+ (calculated for C12H17O4Br: 304.0305 [M]+).

General procedure for synthesis of compounds 13a–13c. To a solution of 1 eq. bromide 12 in 10 mL of CH3CN with stirring, 1.2 eq. was carefully added dropwise piperidine or phenylpiperazine or morpholine and excess K2CO3. The mixture was stirred at 20–25°C for 16 h at room temperature. When the reaction with morpholine was carried out, the reaction mixture was heated (80°C) for 24 h, then a saturated NaCl solution was added and extracted with CHCl3. The combined organic layer was dried with anhydrous Na2SO4, the drying agent was filtered off, and the solvent was distilled off. The resulting product 13a was purified by column chromatography on SiO2 using a hexane–ethyl acetate–methanol mixture as an eluent. Compounds 13b and 13c were used without additional purification.

2-Morpholinoethyl-(1S,4R)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate (13a). Yield 11%, pale yellow crystalline substance, mp 70.6–74.3°С. 1H NMR spectrum (CDCl3), δ, ppm: 0.96 s (3H, 10-Мe), 1.04 s (3H, 9-Мe), 1.08 s (3H, 8-Мe), 1.66 septet (1H, H6endo, J 4.5 Hz), 1.85–2.05 m (2H, H5endo, H6exo), 2.36–2.50 m (5H, H5exo, 2H14, 2H17), 2.60 t. d (2H, H13, 2J 5.7, J 2.7 Hz), 3.64 t (4H, 2H15, 2H16, J 4.7 Hz), 4.34 t (2H, H12, J 5.7 Hz). 13С NMR spectrum (CDCl3), δС, ppm: 178.0 (C3), 167.2 (C11), 90.9 (C7), 66.8 (C15, C16), 61.6 (C12), 56.9 (C13), 54.7 (C1), 54.1 (C4), 53.4 (С14, C17), 30.4 (С6), 28.7 (C5), 16.5 (8-Мe), 16.5 (9-Мe), 9.5 (10-Мe). Mass spectrum (HRMS), m/z: 311.1724 [M]+ (calculated for C16H25O5N: 311.1727 [M]+).

2-(Piperidin-1-yl)ethyl-(1S,4R)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate (13b). Yield 21%, brown solid substance, mp 45.9–48.5°C. 1H NMR spectrum (CDCl3), δ, ppm: 0.97 s (3H, 10-Мe), 1.05 s (3H, 8-Мe), 1.09 s (3H, 9-Мe), 1.34–1.44 m (2H, H16), 1.49–1.55 m (4H, 2H15, 2H17), 1.62–1.70 m (1H, H6endo), 1.86–1.94 m (1H, H5endo), 2.00 septet (1H, H6exo, J 4.6 Hz), 2.37–2.46 m (5H, H5exo, 2H14, 2H18), 2.59 t. d (2H, H13, 2J 6.0, J 2.3 Hz), 4.34 t (2H, H12, J 5.9 Hz). 13С NMR spectrum (CDCl3), δС, ppm: 178.1 (C3), 167.2 (C11), 91.1 (C7), 62.3 (C12), 56.9 (C13), 54.7 (C1), 54.5 (C14, C18), 54.1 (С4), 30.5 (С6), 28.8 (C5), 25.6 (C15, C17), 23.9 (C16), 16.6 (8-Me, 9-Me), 9.6 (10-Мe). Mass spectrum (HRMS), m/z: 309.1932 [M]+ (calculated for C17H27O4N: 309.1935 [M]+).

2-(4-Phenylpiperazin-1-yl)ethyl-(1S,4R)4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate (13c). Yield 17%, yellow crystalline substance, mp 65.0–72.0°С. 1H NMR spectrum (CDCl3), δ, mp: 0.97 s (3H, Мe10), 1.04 s (3H, 9-Мe), 1.09 s (3H, 8-Мe), 1.67 septet (1H, H6endo, J 4.5 Hz), 1.86–1.95 m (1H, H5endo), 1.97–2.06 m (1H, H6exo), 2.37–2.46 m (1H, H5exo), 2.58–2.71 m (6H, Н13, Н14, Н17), 3.10–3.21 m (4H, Н15, Н16), 4.40 t (2H, H12, J 5.8 Hz), 6.81–6.94 m (3H, Н19, Н23, Н21), 7.21–7.28 m (2H, Н20, Н22). 13С NMR spectrum (CDCl3), δС, ppm: 178.1 (C3), 167.6 (C11), 151.0 (С18), 129.3 (С20, С22), 119.7 (С21), 116.1 (С19, С23), 91.1 (C1), 62.0 (C12), 56.5 (С1), 53.1 (С15, С16), 49.1 (С13), 49.0 (С14, С17), 44.9 (C4), 30.52 (С6), 28.72 (C5), 16.8 (8-Мe, 9-Мe), 9.4 (Мe10). Mass spectrum (HRMS), m/z: 386.2232 [M]+ (calculated for C22H30O4N2: 386.2248 [M]+).

Assessment of the cytopathic effect of the studied substances in cell culture. The toxicity of the compounds was studied by evaluation cell viability using the reduction reaction of the tetrazolium dye MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] by cells in culture, the intensity of which reflects the degree of cell viability as a result of dye reduction by mitochondrial and partially cytoplasmic dehydrogenases.

The test substances in the concentration range of 4–300 μg/mL, dissolved in the cell culture medium, were added to the plate wells in a volume of 200 μL and incubated for 48 h at 37°C in an atmosphere of 5% CO2. After the incubation period, the cells were washed with MEM medium and 100 μL of a solution (0.5 mg/mL) of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in cell medium was added to the plates wells. Cells were incubated at 37°C in an atmosphere of 5% CO2 for 2 h and washed for 5 min with saline. The precipitate was dissolved in 100 μL of DMSO per well, and the optical density was measured using a Multiscan FC plate analyzer (Thermo Scientific) at a wavelength of 540 nm. Based on the data obtained, the 50% cytotoxic concentration (CC50) was calculated, i.e., the concentration of a compound that reduces the optical density in the wells twice compared to control cells without drugs.

Study of the antiviral activity of substances. The test samples in a volume of 100 μL were added to the plates wells with a monolayer of MDCK cells. The plates with cells were incubated in an atmosphere of 5% CO2 at 37°C for 1 h. After that, 0.1 mL of virus (m.o.i. 0.01) in alpha-MEM medium was added to the wells and incubated for 48 h in an atmosphere of 5% CO2 at 37°C. At the end of the incubation period, the cells were washed with MEM and cell viability assays were performed as described above. Based on the data obtained, the value of the 50% inhibitory concentration (IC50) was calculated—the concentration of the compound that led to a 50% decrease in the cytodestructive effect of the virus, and the selectivity index (SI)—the ratio of CC50 to IC50. Compounds were considered active if the SI value was 10 and higher.

Methods of quantum chemical calculations. All calculations were carried out on a cluster supercomputer at the Ufa Institute of Chemistry of the Ufa Federal Research Center of the Russian Academy of Sciences. GAUSSIAN C.16 software was used [15]. To optimize and solve the oscillatory problem, we used the B3LYP method [16, 17] in combination with the 6-311+G(d, p) basis set [18]. Calculations to determine the electronic parameters of the studied compounds were carried out in the gas phase approximation. The B3LYP density functional theory method is a hybrid three-parameter functional that combines calculation speed and acceptable accuracy. The literature describes quantum chemical calculations of geometric structures similar to the studied methods of the DFT theory, in particular using the B3LYP functional [19, 20].