Products of natural origin are considered as valuable renewable raw materials for the development of promising therapeutic agents [16], and the creation of drugs from natural sources, especially from plants, has long been the basis for the treatment of various human diseases, including oncological pathologies. In recent years, there has been an increased interest in sesquiterpene lactones due to their antitumor effect on various tumor cell lines [79]. The biological effect of sesquiterpene lactones is realized by inducing apoptosis, as a result of their influence on the formation of reactive oxygen species, leading to oxidative damage in the cell and triggering the mitochondria-dependent pathway of apoptosis [9]. The cytotoxic effect, as a rule, is characteristic of lactones containing a conjugated double bond in the lactone ring, which facilitates nucleophilic addition reactions. Since various molecules with nucleophilic groups are present in a living cell, they are the targets for the action of sesquiterpene lactones. In this regard, various scientific groups are actively studying the ways of structural modification of such molecules and biological studies of various derivatives are being carried out.

Herein, we reported the synthesis of derivatives of sesquiterpene lactones, dehydrocostus lactone 1 and alantolactone 2, and their cytotoxic activity towards various tumor cell lines. The choice of objects of the study is not accidental, since alantolactone 2 is one of the actively studied eudesmane-type sesquiterpene lactones with a wide spectrum of biological activity, including antitumor effects on neoplastic cells [10]. In addition, for dehydrocostus lactone 1 and some of its derivatives, a rather high cytostatic activity was also found [11, 12].

As the main methodology for the structural modification of the selected lactones, we chose an approach based on the use of the cycloaddition reaction of organic azides to acetylene derivatives. This technique of click chemistry has been widely used in recent years [1315] due to the ease of implementation and almost quantitative yield of the target compounds. In addition, it should be noted that the triazole spacer formed as a result of click reactions is more than just a passive linker. It significantly facilitates binding to biological targets due to hydrogen bonds, and also significantly increases solubility and dipole interactions [16].

Recently, a work has been published demonstrating the successful application of the click chemistry reaction to dehydrocostus lactone 1 [17]. The authors synthesized a series of 1,4-disubstituted-1,2,3-triazole conjugates based on dehydrocostus lactone with moderate cytotoxicity.

Since the click chemistry method involves the use of two synthetic blocks, acetylene and azide, we proposed to use the products of nucleophilic addition of propargylamine to lactones 1 and 2 as acetylene blocks. The reaction was carried out in boiling ethanol for 8 h (Scheme 1).

Scheme
scheme 1

1.

Both propargylated lactones 3 and 4, which are a mixture of diastereoisomers isolated by column chromatography in 58 and 56% yields, respectively, are viscous oils, the structure of which was determined by a complex of spectral methods (1Н, 13С NMR, IR spectroscopy and mass spectrometry), and composition by elemental analysis data.

The most strong arguments confirming the structure of compounds 3 and 4 are the presence of triplet signals in the 1Н NMR spectra in the region of 2.25 ppm with a characteristic spin-spin coupling constant 4JHH 2.4 Hz, as well as the presence in the 13С NMR spectra of two signals of sp-hybridization carbon atoms in the 70– 80 ppm region. Additional confirmation of the structure of compounds 3 and 4 are the data of IR spectroscopy: the presence of weak absorption bands in the 2100 cm–1 region.

As the second azide block, it was proposed to use substituted benzyl azides 6a6d, which were obtained in two stages according to the known procedures [1820] using benzyl alcohols 5a5d as starting compounds (Scheme 2). Azides 6a6d were isolated individually using column chromatography. The physico-chemical characteristics and spectral data for compounds 6a6d fully correspond to the literature data [1820].

Scheme
scheme 2

2.

The reaction of acetylenes 3 and 4 with azides 6a6d was carried out under standard click chemistry conditions (Scheme 3). The reaction progress was monitored by thin layer chromatography.

Scheme
scheme 3

3.

The target products, which are mixtures of diastereoisomers, were isolated by column chromatography on silica gel using a mixture of CHCl3–EtOH (100:0.5→100:10) as an eluent. Their structure was proved by spectral methods (1H and 13C NMR, IR spectroscopy and mass spectrometry), and their composition was proved by analytical data.

The formation of the triazole ring is accompanied by the disappearance of signals from the C≡CH group in the spectra of the products and the appearance of new signals that unambiguously indicate the presence of such a ring. For example, the 1Н NMR spectra of compounds 7 and 8 contain a characteristic downfield singlet signal at 7.39–7.54 ppm of the С16Н proton. The spectra of compounds 7, 8 contain all signals related to the initial acetylenic and azide components, not affected by the formation of a new ring.

Since the main goal of studying the biological activity of the synthesized derivatives of sesquiterpene lactones was to study their antitumor activity, it was necessary to determine their cytotoxic profile, since one of the key properties of potential antineoplastic agents for the treatment of oncopathologies is the presence of cytotoxicity towards tumor cells. The effect of new derivatives of sesquiterpene lactones on the survival of cell cultures was determined by the IC50 value in relation to a number of tumor cells of the lines A549, SH-SY5Y, Hep-2 and HeLa. Viability was examined using the MTT test. As can be seen from Table 1, compound 7b exhibits the most pronounced toxic effect with respect to all cell cultures, as evidenced by the IC50 values of the cytotoxic effect, which do not exceed 26 μM. In turn, compounds 7a and 7c exhibited similar activity on HeLa and SH-SY5Y cell lines, but without a pronounced effect on the viability of Hep-2 and A549 cells. For derivatives 8a8c, no cytotoxic effect was found on any of the cell lines in the selected concentration range. It should be noted that all compounds, except for derivative 7b, had no effect on the Hep-2 line.

Table 1. Cytotoxicity of sesquiterpene lactone derivatives towards tumor cell lines

Summarizing the data in Table 1, we can conclude that derivatives 7a7c exhibit the highest cytotoxic activity, containing in their structure a fragment of dihydrocostus lactone, a natural sesquiterpene lactone obtained from various medicinal plants, such as Inula helenium L. and Saussurea lappa [21]. Studies by other authors have shown that dehydrocostus lactone has antitumor properties associated with reduced NF-kB activity [22], is able to induce both internal and external signaling pathways of apoptosis by activating caspase 9 [23] and 8, respectively, and also activates the effector caspase 3 [24], leading to DNA fragmentation.

In conclusion, using the click chemistry methodology, we obtained a number of previously undescribed conjugates of alantolactone and dehydrocostus lactone with aromatic alkoxy derivatives. It was found that dehydrocostus lactone derivatives showed the greatest cytotoxic effect towards A549, SH-SY5Y, Hep-2, and HeLa tumor cells.

EXPERIMENTAL

Chemically pure organic solvents were dehydrated and purified according to standard procedures. CDCl3 solvent (Acros) was used without further purification.

1Н and 13С{1Н} NMR spectra were recorded on a Bruker AV-400 instrument in CDCl3 solutions using the residual proton signal of the deuterated solvent as an internal standard (1Н, 13С). The 13C NMR spectra were recorded in the JMODECHO mode; the signals of carbon atoms with an even and odd number of protons have opposite polarities. IR spectra were recorded on a Magna-IR 750 Nicolet IR Fourier spectrometer (thin layer). The reaction progress was monitored by TLC on Alumina TLC Plates w/UV254. Chromatographic purification of substances was carried out on Macherey-Nagel silica gel (MN Kieselgel 60, 70–230 mesh). Elemental analysis was carried out at the Microanalysis Laboratory of the A.N. Nesmeyanov Institute of Organoelement Compounds of RAS. Analysis by liquid chromatography–mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 instrument (Japan) using electrospray ionization (ESI). HPLC grade acetonitrile was used as the mobile phase.

Alantolactone and dehydrocostus lactone were isolated from plant substrates according to previously reported procedures [25, 26].

General procedure for the propargylation of dehydrocostus lactone 1 and alantolactone 2. The propargylation of lactones 1 and 2 was carried out in accordance with the previously described procedure [27]. A mixture of lactone 1 or 2 (0.5 mmol) and propargylamine (55 mg, 1 mmol) in EtOH (5.0 mL) was refluxed for 8 h. After cooling, the solvent was removed in vacuum. The residue was purified using silica gel column chromatography, eluent—petroleum ether–AcOEt (3 : 1→1 : 1). After removal of the solvent, the residue was dried in vacuum (0.1 mmHg).

6,9-Dimethylene-3-[(prop-2-ylamino)methyl]decahydroazuleno[4,5-b]furan-2(9bH)-one (3). Yield 82 mg (58%), diastereomers mixture, colorless oil. IR spectrum, ν, cm–1: 650, 895, 999, 1112, 1129, 1176, 1207, 1320, 1337, 1442, 1460, 1641, 1766 (C=O), 2856, 2926, 3290. 1H NMR spectrum (СDCl3), δ, ppm: 2.15–1.80 m and 1.37–1.25 m (6H, CH-terpene ring), 2.25 t (1H, C18H, 4JНН 2.4 Hz), 2.60–2.40 m and 2.35–2.25 m (5H, CH-terpene ring), 3.05–2.80 m (4H, C15H2 + C12H + C13H), 3.46 d (2H, C16H2 , 4JНН 2.4 Hz), 3.97 t (1H, C2H, 3JНН 9.2 Hz), 4.78 s (1H, C14HВ), 4.88 s (1H, C14HA), 5.05 d (1H, C13HB, 4JНН 1.6 Hz), 5.18 d (1H, C13HA, 4JНН 1.6, C13HA). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 30.00 (C8H2), 32.35 (C4H2), 32.38 (C5H2), 37.45 (C9H2), 38.41 (C15H2), 44.95 (C3H), 46.42 (C16H2), 46.84 (C7H), 47.19 (C1H), 51.66 (C12H), 71.69 (C18H), 81.33 (C17), 85.63 (C2H), 109.00 (C14H2), 111.76 (C13H2), 149.64 (C6), 151.51 (C10), 177.45 (C11=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 286.000 (7.66) [M + H]+, 593.150 (2.60) [2M + Na]+. Found, %: C 75.51; H 8.13; N 4.73. C18H23NO2. Calculated, %: C 75.76; H 8.12; N 4.91.

(5S,8aR)-5,8a-Dimethyl-3-(prop-2-ylamino)methyl-3,3a,6,7,8,8a,9,9a-octahydronaphtho[2,3-b]furan-2(5H)-one (4). Yield 80 mg (56%), diastereomers mixture, colorless oil. IR spectrum, ν, cm–1: 668, 895, 947, 971, 1000, 1038, 1120, 1150, 1181, 1210, 1326, 1338, 1376, 1458, 1687, 1759 (C=O), 2868, 2928, 3289, 3307. 1H NMR spectrum (СDCl3), δ, ppm: 1.22 s (3H, C13H3), 1.11 d (3H, C14H3, 3JНН 8.0 Hz), 2.12–2.04 м, 1.83–1.80 m and 1.61–1.40 m (9H, CH-terpene ring), 2.24 t (1H, C18H, 4JНН 3.8 Hz), 3.14–2.98 m, 2.85–2.81 m and 2.49–2.46 m (5H, C15H2 + CH-terpene ring), 3.47 d (2H, C16H2, 4JНН 3.8 Hz), 4.76–4.74 m (1H, C9H), 5.18 d (1H, C7H, 3JНН 3.0 Hz). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 16.62 (C1H2), 22.71 (C13H3), 28.45 (C14H3), 32.62 (C2H2), 32.79 (C6H2), 37.43 (C10H2), 38.18 (C8H), 38.24 (C3H), 41.96 (C5), 42.50 (C15H2), 45.28 (C16H2), 45.46 (C11H), 71.63 (C18H), 77.20 (C9H), 81.37 (C17), 114.81 (C7H), 150.87 (C4), 177.56 (C12=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 288.050 (7.75) [M + H]+. Found, %: C 67.90; H 7.01; N 4.42. 2C18H25NO2·CH2Cl2. Calculated, %: C 67.36; H 7.94; N 4.25.

General procedure for the synthesis of triazoles 7a8d and 8a8d. A mixture of acetylene 3 or 4 (0.5 mmol), t-BuOH (5 mL), H2O (2 mL), CuSO4·5H2O (5 mol%) was stirred for 10 min, then sodium ascorbate (~20 mg) and the corresponding azide 6 (0.5 mmol) was added with stirring. The resulting mixture was stirred for 24 h. After completion of the reaction, the solvent was removed in vacuum and DCM (10 mL) was added to the residue. The organic layer was separated, DCM was removed in vacuum, the residue was chromatographed on silica gel (eluent—CHCl3–EtOH, 100 : 0.5→100 : 10). After removing the solvent, the residue was kept in vacuum (0.1 mmHg) for 5 h.

3-({[1-(Benzo[d][1,3]dioxol-5-ylmethyl)-1H-1,2,3-triazol-4-yl]methylamino}methyl)-6,9-dimethylenenedecahydroazuleno[4,5-b]furan-2(9bH)-one (7a). Yield 81%, diastereomers mixture, yellow oil. IR spectrum, ν, cm–1: 515, 683, 775, 896, 927, 997, 1038, 1177, 1210, 1249, 1336, 1337, 1447, 1492, 1504, 1641, 1765 (C=O), 2857, 3080. 1H NMR spectrum (СDCl3), δ, ppm: 1.25–1.37 m, 1.80–2.10 m, 2.19–2.28 m, 2.40–2.55 m, 2.79–2.97 m (14H, CH-terpene ring + C24H2), 3.92 s (2H, C25H2), 3.95 t (1H, C2H, 3JНН 8.0), 4.76 br. s, 4.86 br. s, 5.03 br. s and 5.16 br. s (4H, C14H2 + C13H2), 5.40 s (2H, C23H2), 5.96 s (2H, OCH2O), 6.75 s (1H, C17H), 6.79 br. s (2H, C21H + C20H), 7.45 s (1H, C16H). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 29.98 (C4H2), 32.37 (C9H2 + C8H2), 37.40 (C5H2), 44.64 (C12H), 44.93 (C25H2), 46.80 (C1H), 46.83 (C24H2), 47.00 (C7H), 51.64 (C3H), 53.80 (C23H2), 85.66 (C2H), 101.23 (OCH2O), 108.41 (C17H), 108.93 (C14H2), 111.73 (C13H2), 121.39 (C21H), 121.81 (C20H), 128.09 (C22), 146.52 (C15), 147.85 (C6), 148.10 (C10), 149.60 (C19), 151.53 (C18), 177.63 (C11=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 463.050 (10.28) [M + H]+, 485.100 (79.00) [М + Na]+. Found, %: C 61.07; H 5.81; N 10.27. 1.25C26H30N4O4·CH2Cl2. Calculated, %: C 60.68; H 6.00; N 10.56.

3-({[1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl]methylamino}methyl)-6,9-dimethylenedecahydroazuleno[4,5-b]furan-2(9bH)-one (7b). Yield 78%, diastereomers mixture, yellow oil. IR spectrum, ν, cm–1: 514, 666, 755, 895, 998, 1033, 1125, 1178, 1210, 1250, 1335, 1441, 1462, 1515, 1613, 1641, 1766 (C=O), 2855, 2932, 3080. 1H NMR spectrum (СDCl3), δ, ppm: 1.24–1.34 m, 1.82–2.05 m, 2.17–2.24 m, 2.34–2.51 m, 2.75–2.94 m (14H, CH-terpene ring + C24H2), 3.78 s and 3.88 s (5H, C25H2 + OCH3), 3.92 t (1H, C2H, 3JНН 8.0 Hz), 4.74 br. s, 4.84 br. s, 5.01 br. s, 5.15 br. s (4H, C14H2 + C13H2), 5.42 s (2H, C23H2), 6.87 d (2H, C17H + C21H, 3JНН 8.0 Hz), 7.21 d (2H, C18H + C20H, 3JНН 8.0 Hz), 7.39 s (1H, C16H). 13C{1H} NMR spectrum (CDCl3) δС, ppm: 29.91 (C4H2), 32.38 (C9H2 + C8H2), 37.37 (C5H2), 44.66 (C25H2), 44.80 (C1H), 46.72 (C12H), 46.79 (C24H2), 47.05 (C7H), 51.58 (C3H), 53.40 (C23H2), 55.08 (OCH3), 85.54 (C2H), 108.83 (C14H2), 111.63 (C13H2), 114.18 (C17H + C21H), 121.17 (C16H), 126.43 (C22), 129.44 (C18H + C20H), 146.63 (C15), 149.55 (C6), 151.50 (C10), 159.61 (C19), 177.56 (C11=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 449.150 (11.82) [M + H]+, 471.050 (72.46) [М + Na]+, 919.250 (100.00) [2M + Na]+. Found, %: C 64.46; H 6.51; N 11.29. 1.75C26H32N4O3·CH2Cl2. Calculated, %: C 64.20; H 6.72; N 11.27.

3-[{(1-(3,4-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl]methylamino}methyl)-6,9-dimethylenedecahydroazuleno[4,5-b]furan-2(9bH)-one (7c). Yield 80%, diastereomers mixture, yellow oil. IR spectrum, ν, cm–1: 555, 666, 753, 894, 998, 1026, 1048, 1142, 1160, 1240, 1263, 1336, 1441, 1464, 1517, 1594, 1641, 1764 (C=O), 2853, 2931, 3080. 1H NMR spectrum (СDCl3), δ, ppm: 1.21–1.37 m, 1.80–2.05 m, 2.19–2.27 m, 2.38–2.55 m, 2.76–2.96 m (14H, CH-terpene ring + C24H2), 3.85 s and 3.88 s (6H, OCH3), 3.93 s (2H, C25H2), 3.95 t (1H, C2H 3JНН 8.0 Hz), 4.77 br. s, 4.87 br. s, 5.03 br. s, 5.16 br. s (4H, C14H2 + C13H2), 5.44 s (2H, C23H2), 6.81–6.88 m (3H, C21H + C20H + C17H), 7.45 s (1H, C16H). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 30.00 (C4H2), 32.36 (C9H2 + C8H2), 37.38 (C5H2), 44.67 (C25H2), 44.98 (C3H), 46.87 (C24H2), 47.02 (C1H), 51.68 (C7H), 53.92 (C23H2), 55.82 and 55.79 (OCH3), 85.66 (C2H), 108.98 (C14H2), 111.08 (C20H), 111.12 (C21H), 111.79 (C13H2), 120.74 (C17H), 121.42 (C16H), 126.85 (C22), 146.50 (C6), 149.27(C10), 149.58 (C19), 151.52 (C18), 177.64 (C11=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 479.150 (13.64) [M + H]+, 501.100 (68.57) [М + Na]+, 979.300 (100.00) [2M + Na]+. Found, %: C 61.67; H 6.66; N 10.02. 1.25C27H34N4O4·CH2Cl2. Calculated, %: C 61.09; H 6.57; N 10.25.

3-({[1-(2,3-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl]methylamino}methyl)-6,9-dimethylenedecahydroazuleno[4,5-b]furan-2(9bH)-one (7d). Yield 82%, diastereomers mixture, yellow oil. IR spectrum, ν, cm–1: 502, 667, 754, 894, 1002, 1048, 1076, 1176, 1227, 1272, 1433, 1485, 1589, 1641, 1699, 1766 (C=O), 2854, 2934, 3081. 1H NMR spectrum (СDCl3), δ, ppm: 1.27–1.33 m, 1.92–2.05 m, 2.44–2.52 m, 2.80–2.95 m (14H, CH-terpene ring + C24H2), 3.84 s and 3.88 s (6H, OCH3), 3.93 s (2H, C25H2), 3.95 t (1H, C2H, 3JНН 8.0 Hz), 4.77 br. s, 4.87 br. s, 5.05 br. s, 5.18 br. s (4H, C14H2 + C13H2), 5.54 br. s (3H, C23H2 + NH), 6.83 d (1H, C19H, 3JНН 8.0 Hz), 6.93 d (1H, C21H, 3JНН 8.0 Hz), 7.04 t (1H, C20H 3JНН 8.0 Hz), 7.54 s (1H, C16H). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 30.00 (C4H2), 31.43 (C8H2), 32.37 (C9H2), 37.39 (C5H2), 44.36 (C25H2), 45.09 (C12H), 46.66 (C24H2), 46.83 (C7H), 48.86 (C23H2), 51.66 (C3H), 55.66 and 60.75 (2 OCH3); 85.82 (C2H), 108.99 (C14H2), 111.80 (C13H2), 113.02 (C19H), 121.51 (C20H), 122.48 (C16H), 124.32 (C21H), 128.14 (C22), 145.07 (C15), 146.86 (C6), 149.59 (C10), 151.52 (C17), 151.73 (C18), 177.58 (C11=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 479.100 (13.38) [M + H]+, 501.100 (92.53) [М + Na]+, 979.300 (100.00) [2M + Na]+. Found, %: s 57.37; H 6.24; N 9.02. C27H34N4O4·1.4·CH2Cl2. Calculated, %: s 57.09; H 6.21; N 9.38.

(5S,8aR)-3-{[(1-(Benzo[d][1,3]dioxol-5-ylmethyl)-1H-1,2,3-triazol-4-yl]methylamino}methyl)-5,8a-dimethyl-3 ,3a,6,7,8,8a,9,9a-octahydronaphtho[2,3-b]furan-2(5H)-one (8a). Yield 78%, diastereomers mixture, yellow oil. IR spectrum, ν, cm–1: 518, 669, 775, 811, 927, 1120, 1039, 1149, 1181, 1216, 1249, 1339, 1376, 1447, 1492, 1504, 1758 (С=О), 2929, 3139. 1H NMR spectrum (СDCl3), δ, ppm : 1.08 d (3H, C14H3 3JНН 8.0 Hz), 1.12 s (3H, C13H3), 1.07–1.12 m, 1.39–159 m, 1.74–1.85 m, 2.05–2.10 m, 2.35–2.50 m, 2.75–2.85 m, 2.95–3.05 m, 3.09–3.11 m (14H, C24H2 + CH-terpene ring), 3.98 d (1H, C25HB 2JНН 12.0 Hz), 3.94 d (1H, C25HA, 2JНН 12.0 Hz), 4.71–4.73 m (1H, C9H), 5.10 d (1H, C7H, 3JНН 4.0 Hz), 5.39 s (2H, C23H2), 5.96 s (2H, OCH2O), 6.74 s (1H, C20H), 6.78 br. s (2H, C17H + C21H), 7.42 s (1H, C16H). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 16.62 (C1H2), 22.72 (C14H3), 28.45 (C13H3), 32.61 (C2H2), 32.78 (C6H2), 37.41 (C8H), 38.17 (C3H), 41.97 (C25H2), 42.47 (C10H2), 44.59 (C24H2), 45.49 (C11H), 45.73 (C23H2), 53.76 (C5), 77.21 (C9H), 101.20 (OCH2O), 108.37 (C21H), 108.41 (C20H), 114.73 (C7H), 121.27 (C16H), 121.80 (C17H), 128.01 (C22), 147.82 (C15), 148.07 (C4), 146.56 and 150.87 (C18 + C19), 177.67 (C12=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 465.100 (15.06) [M + H]+, 487.100 (100.00) [М + Na]+, 951.250 (96.51) [2M + Na]+. Found, %: C 60.78; H 6.38; N 10.76. 1.3C26H32N4O4·CH2Cl2. Calculated, %: C 60.82; H 6.39; N 10.61.

(5S,8aR)-3-({[1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl]methylamino}methyl)-5,8a-dimethyl-3,3a,6,7,8,8a, 9,9a-octahydronaphtho[2,3-b]furan-2(5H)-one (8b). Yield 80%, diastereomers mixture, yellow oil. IR spectrum, ν, cm–1:516, 554, 636, 669, 703, 786, 824, 896, 972, 1000, 1036, 1126, 1149, 1179, 1213, 1250, 1326, 1376, 1442, 1464, 1515, 1586, 1613, 1649, 1760 (C=O), 2868, 2929, 3137. 1H NMR spectrum (СDCl3), δ, ppm: 1.07 d (3H, C14H3 3JНН 8.0 Hz), 1.08–1.12 m (1H, CH-terpene ring), 1.20 s (3H, C13H3), 1.35–1.85 m, 2.05–2.20 m, 2.40–2.45 m, 2.77–3.15 m (10H, CH-terpene ring), 3.79 s (3H, OCH3), 3.90 br. s (1H, NH), 3.92 d. t (1H, C9H, 3JНН 12.0, C9H), 4.73– 5.10 br. s (2H, C25HA + C25HB), 5.43 br. s (2H, C23H2), 6.88 d (2H, C17H + C21H, 3JНН 8.0 Hz), 7.22 d (2H, C18H + C20H, 3JНН 8.0 Hz), 7.40 s (1H, C16H). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 16.22 (C1H2), 22.73 (C14H3), 28.45 (C13H3), 32.62 (C2H2), 32.79 (C6H2), 37.40 (C8H), 38.17 (C3H), 41.97 (C25H2), 42.47 (C10H2), 44.56 (C24H2), 45.45 (C11H), 45.68 (C23H2), 53.47 (C5), 55.12 (OCH3), 77.22 (C9H), 114.24 (C17H + C21H), 114.71 (C7H), 121.30 (C16H), 126.39 (C22), 129.50 (C18H + C20H), 146.35 (C15), 150.88 (C4), 159.68 (C19), 177.65 (C12=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 451.150 (14.29) [M + H]+, 473.100 (93.93) [М + Na]+, 923.350 (100.00) [2M + Na]+. Found, %: C 63.64; H 6.59; N 10.91. 1.5C26H34N4O3·CH2Cl2. Calculated, %: C 63.15; H 7.02; N 11.05.

(5S,8aR)-3-({[1-(3,4-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl]methylamino}methyl)-5,8a-dimethyl-3,3a,6,7,8, 8a,9,9a-octahydronaphtho[2,3-b]furan-2(5H)-one (8c). Yield 82%, diastereomers mixture, yellow. IR spectrum, ν, cm–1: 556, 636, 667, 755, 860, 877, 970, 1027, 1143, 1160, 1180, 1241, 1264, 1325, 1376, 1422, 1442, 1465, 1518, 1594, 1608, 1758 (C=O), 2868, 2930, 3138. 1H NMR spectrum (СDCl3), δ, ppm: 1.07 d (3H, C14H3, 3JНН 8.0 Hz), 1.09–1.13 m (1H, CH-terpene ring), 1.20 s (3H, C13H3), 1.40–1.85 m, 2.06–2.11 m, 2.35–2.50 m, 2.75–3.15 m (10H, CH-terpene ring), 3.84 s and 3.87 s (6H, OCH3), 3.93 d. t (1H, C9H, 3JНН 12.0 Hz), 4.74 br. s and 5.11 br. s (2H, C25HA + C25HB), 5.43 br. s (2H, C23H2), 6.80–6.90 m (2H, C21H + C17H), 7.43 s (1H, C16H). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 16.63 (C1H2), 22.74 (C14H3), 28.47 (C13H3), 32.64 (C2H2), 32.81 (C6H2), 37.45 (C8H), 38.21 (C3H), 41.99 (C25H2), 42.49 (C10H2), 44.58 (C24H2), 45.44 (C11H), 45.72 (C23H2), 53.88 (C5), 55.76 and 55.78 (2 OCH3), 77.27 (C9H), 111.09 (C7H), 114.69 (C20H), 120.72 (C21H), 121.40 (C16H), 126.75 (C22), 146.37 (C4), 149.24 (C19), 150.98 (C18), 177.72 (C12=O). Mass spectrum (LC-MS ESI), m/z (Irel, %): 481.200 (13.80) [M + H]+, 503.150 (87.79) [М + Na]+, 983.350 (100.00) [2M + Na]+. Found, %: C 60.28; H 7.09; N 9.69. 1.25C27Н36N4O4·CH2Cl2. Calculated, %: C 60.87; H 6.91; N 10.21.

(5S,8aR)-3-({[1-(2,3-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl]methylamino}methyl)-5,8a-dimethyl-3,3a,6,7,8, 8a,9,9a-octahydronaphtho[2,3-b]furan-2(5H)-one (8d). Yield 85%, diastereomers mixture, yellow oil. IR spectrum, ν, cm–1: 636, 337, 754, 896, 1003, 1048, 1077, 1125, 1149, 1178, 1228, 1273, 1325, 1377, 1432, 1465, 1486, 1589, 1695, 1760 (С=О), 2868, 2930, 3142. 1H NMR spectrum (СDCl3), δ, ppm: 1.08–1.7 m, 2.05–2.12 m, 2.35–2.46 m (18H, CH-terpene ring), 2.82–3.13 m (3H, C8H + C24H2), 3.85 s, 3.88 s (6H, OCH3), 4.75 br. s and 5.13 br. s (2H, C25HA + C25HB), 5.54 br. s (3H, C7H + C23H2), 6.83 d (1H, C21H, 3JНН 8.0 Hz), 6.93 d (1H, C19H, 3JНН 8.0 Hz), 7.05 t (1H, C20H, 3JНН 8.0 Hz), 7.54 s (1H, C16H). 13C{1H} NMR spectrum (CDCl3,) δС, ppm: 14.40 (C14H3), 16.69 (C1H2), 22.79 (C13H3), 28.51 (C3H), 32.70 (C25H2), 32.85 (C10H2), 37.46 (C8H), 38.23 (C11H), 42.06 (C24H2), 42.56 (C23H2), 48.85 (C5), 55.66.74 and 60.74 (2 OCH3), 77.29 (C9H), 113.06 (C19H), 114.75 (C7H), 121.46 (C16H), 121.56 (C20H), 124.31 (C21H), 128.15 (C22), 146.95 (C4), 150.99 (C17), 152.60 (C18), 177.62 (C12=O). Mass spectrum LC-MS (ESI), m/z (Irel, %): 481.100 (37.73) [M + H]+, 503.100 (100.00) [М + Na]+, 983.250 (81.94) [2M + Na]+. Found, %: C 55.36; H 6.18; N 9.39. C27Н36N4O4·1.6CH2Cl2. Calculated, %: C 55.72; H 6.41; N 9.09.

Study of the cytotoxic profile of sesquiterpene lactone derivatives. Cell cultivation. Cell cultures of tumor lines A549 (pulmonary adenocarcinoma), SH-SY5Y (neuroblastoma), Hep-2 (epidermoid carcinoma of the larynx), HeLa (cervical carcinoma) were provided by the Laboratory of Tumor Cell Genetics of the N.N. Blokhin National Medical Research Center of Oncology and Institute of Cytology of RAS. Cells were grown in DMEM (Gibco, UK) containing fetal bovine serum (10% v/v) (ThermoFisher Scientific, UK), L-glutamine (2 mM) (Gibco, UK) and penicillin-streptomycin (1% v/v) (PanEco, Russia) at 37°C in a humidified CO2 atmosphere (5%). Upon reaching 80% confluence, the cells were passaged using a 0.25% trypsin-EDTA solution (PanEco, Russia).

Cell viability assay. Cell viability was determined using the MTT assay [27]. The method is based on the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by mitochondrial dehydrogenases of living cells to crystals of insoluble formazan, thus, the amount of formazan formed reflects cell viability. Each cell line was seeded in 96-well plates (1×104 cells/well) and cultured for 24 h at 37°C under CO2 (5%) atmosphere. Incubation with test substances at selected concentrations (0.1, 1, 10, 30, 100 μM) was carried out for 24 h, then MTT solution (5 mg/mL in 0.9% NaCl) was added to each well, cells were incubated for 2 h at 37°C. After removal of the nutrient medium, dimethyl sulfoxide was added to each well to dissolve the formazan. Using a Victor 3 plate analyzer (PerkinElmer), the absorbance at 530 nm was determined minus the measured background absorbance at 620 nm. The concentration value causing 50% inhibition of cell population growth (IC50) was determined from dose-response curves using GraphPad Prism 9.0 software.