Isolation, Structure Determination of Sesquiterpenes from Neurolaena lobata and Their Antiproliferative, Cell Cycle Arrest-Inducing and Anti-Invasive Properties against Human Cervical Tumor Cells

Seven new germacranolides (1–3, 5–8), among them a heterodimer (7), and known germacranolide (4), eudesmane (9) and isodaucane (10) sesquiterpenes were isolated from the aerial parts of Neurolaena lobata. Their structures were determined by using a combination of different spectroscopic methods, including HR-ESIMS and 1D and 2D NMR techniques supported by DFT-NMR calculations. The enantiomeric purity of the new compounds was investigated by chiral HPLC analysis, while their absolute configurations were determined by TDDFT-ECD and OR calculations. Due to the conformationally flexible macrocycles and difficulties in assigning the relative configuration, 13C and 1H NMR chemical shift and ECD and OR calculations were performed on several stereoisomers of two derivatives. The isolated compounds (1–10) were shown to have noteworthy antiproliferative activities against three human cervical tumor cell line with different HPV status (HeLa, SiHa and C33A). Additionally, lobatolide C (6) exhibited substantial antiproliferative properties, antimigratory effect, and it induced cell cycle disturbance in SiHa cells.


Introduction
Sesquiterpene lactones (SLs) are one of the most prevalent and biologically significant classes of secondary metabolites of plants, comprising over 5000 known compounds. They are common in several families (e.g., Apiaceae, Solanaceae, Cactaceae and Euphorbiaceae), but the majority of them have been obtained from Asteraceae [1,2]. These lactones are classified biogenetically, according to the carbocyclic skeleton into four main groups: germacranolides, eudesmanolides, guaianolides and pseudoguaianolides. Besides these main types, there are a varieties of other lactones, formed by further modification of the carbon skeleton during biosynthesis. Germacranolides are considered as progenitors for other  53 45 W) in San José, Guatemala. Voucher specimens were archived at the herbarium of the Institute for Ethnobiology, San Jose, Guatemala, and at the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary (No. 813). The fresh plant material (aerial parts) was air-dried and stored deep-frozen until subsequent extraction.

Computational Section
Mixed torsional/low-mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the MMFF with an implicit solvent model for CHCl 3 applying a 21 kJ/mol energy window [24]. Geometry re-optimizations of the resultant con- and DFT-NMR calculations [mPW1PW91/6-311+G(2d,p) and mPW1PW91/6-311+G(2d,p) SMD/CHCl 3 ] were performed with the Gaussian 09 package [25]. ECD spectra were generated as sums of Gaussians with 2400-3600 cm −1 width at half-height, using dipole-velocity-computed rotational strength values [26]. Computed NMR shift data were corrected with I = 185.4855 and S = −1.0306 for the carbons and I = 31.8996 and S = −1.0734 for the hydrogens in the gas-phase and I = 186.5242 and S = −1.0533 for the carbons and I = 31.8018 and S = −1.0936 for the hydrogens in the SMD calculations [27,28]. Boltzmann distributions were estimated from the CAM-B3LYP and the B3LYP energies. Visualization of the results was performed by the MOLEKEL software package [29].

Antiproliferative MTT Assay
Antiproliferative effect of the isolated compounds were determined in vitro using SiHa (HPV 16+), HeLa (HPV 18+), C33A (HPV negative) human cervical cell lines, NIH-3T3 mouse embryonic and MRC-5 human fibroblast cells by means of MTT ([3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay. Briefly, a limited number of human cancer cells (5000/well for SiHa and HeLa cells, 10,000/well in case of C33A cells) were seeded onto a 96-well microplate and became attached to the bottom of the well overnight. On the second day of the procedure, the test substances were added in two concentrations (10.0 and 30.0 µM) in order to obtain preliminary data and then the compounds were applied in serial dilution (applied final concentrations were: 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 µM). Further details of the experiment were described in ref. [30]. All in vitro experiments were carried out on two 96-well dishes with at least five parallel wells [31].

Cell Cycle Analysis by Flow Cytometry
Cell cycle analysis was performed as described in [32,33].

Wound Healing Assay
In order to assess antimetastatic activity of the tested compound, wound healing assay was performed. The assay was done with specific wound healing assay chambers (Ibidi GmbH, Gräfelfing, Germany). SiHa cells were collected and 35,000 cells were seeded into both chambers of the insert. Cells were let to be attached to the plate surface during an overnight incubation at 37 • C in 5% CO 2 atmosphere and then the inserts were removed. Cell debris was removed by a washing step with PBS. Test compounds were added to the wells in increasing concentrations in 2% FBS containing medium for 24 and 48 h. Migration of the cells into the wound site was visualized by phase-contrast inverted microscope (Axiovert 40, Zeiss, Thornwood, NY, USA). Images were taken with CCD camera at definite intervals and the migration of the cells was calculated as the ratio of wound closure by ImageJ software [34].

Statistical Analysis
Statistical analysis of the obtained data was performed by analysis of variance (ANOVA) followed by Dunnett's test. All analyses were performed with GraphPad Prism 5 (Graph-Pad Software; San Diego, CA, USA).

Results and Discussion
Phytochemical investigation of the CH 2 Cl 2 -soluble phase of the MeOH extract, obtained from the aerial parts of N. lobata, resulted in the isolation of ten sesquiterpenes (1-10) (Figure 1). For the separation of the compounds a combination of different chromatographic methods, including open column chromatography (OCC), vacuum liquid chromatography (VLC), rotation planar chromatography (RPC), and preparative TLC were used. The structure elucidation was carried out by extensive spectroscopic analysis, using HR-ESIMS, and 1D and 2D NMR ( 1 H-1 H COSY, HSQC, HMBC and NOESY) spectroscopy and DFT-NMR calculations.  , were attributed to an isovaleroyloxy group. The chemical shifts and coupling constants of 1 were closely related to those of the 1-keto-furanoheliangolide derivative 8-isovaleroyloxy-9α-hydroxy-calyculatolide [9], with the only difference changing of the positions of isovaleroyloxy and hydroxy groups. The isovaleroyl group was assigned to C-9 with regard to the HMBC correlations between H-9 and the carbonyl carbon signal of isovaleroyl group. The relative configuration of compound 1 was determined by means of a NOESY experiment. The coupling constant of H-6 and H-7 (J 6,7 = 5.0 Hz) indicated the β orientation of H-6 and α orientation of H-7, found in all sesquiterpenes isolated previously from this plant. NOESY correlation between H-6 and H-15 confirmed the β position of the 15-methyl group, while NOE effects observed between H-7 and H-5a, H-5a and H-4, H-7 and H-13b, and H-13b and H-8 dictated the α orientation of these protons. All of the above evidence supported the structure of this compound as 8β-hydroxy-9α-isovaleroyloxycalyculatolide, and named as lobatolide A (1).

Statistical Analysis
Statistical analysis of the obtained data was performed by analysis of variance (ANOVA) followed by Dunnett's test. All analyses were performed with GraphPad Prism 5 (GraphPad Software; San Diego, CA, USA).

Results and Discussion
Phytochemical investigation of the CH2Cl2-soluble phase of the MeOH extract, obtained from the aerial parts of N. lobata, resulted in the isolation of ten sesquiterpenes (1-10) (Figure 1). For the separation of the compounds a combination of different chromatographic methods, including open column chromatography (OCC), vacuum liquid chromatography (VLC), rotation planar chromatography (RPC), and preparative TLC were used. The structure elucidation was carried out by extensive spectroscopic analysis, using HR-ESIMS, and 1D and 2D NMR ( 1 H-1 H COSY, HSQC, HMBC and NOESY) spectroscopy and DFT-NMR calculations.  The absolute configuration of 1 was determined by the solution TDDFT-ECD method [35,36]. Merck Molecular Force Field (MMFF) conformational search of (4R,6R,7R,8S,9R,10R)-1 resulted in 133 initial conformers in a 21 kJ/mol energy window. These structures were re-optimized at the CAM-B3LYP/TZVP [37] PCM/MeCN levels yielding 12 low-energy conformers over 1% Boltzmann population. ECD spectra computed at various levels of theory for these conformers reproduced well the experimental ECD spectrum ( Figure 2). Furthermore, the low-energy conformers, differing only in the orientation of the OiVal and the OH groups (Figure 3), gave similar computed ECD spectra allowing the solid elucidation of the absolute configuration as (4R,6R,7R,8S,9R,10R)-1. To test the applicability of the DFT-NMR method [28,38], the 133 MMFF conformers were also re-optimized at the B3LYP/6-31+G(d,p) level yielding seven low-energy conformers. The NMR shift values were computed for these conformers both at the mPW1PW91/6-311+G(2d,p) and the mPW1PW91/6-311+G(2d,p) SMD/CDCl 3 levels [39]. Computed and experimental 13 C NMR chemical shift values gave a good overall agreement with CMAE (corrected mean absolute error) values [40] of 1.92 and 1.77 ppm, respectively (Table S1). Although the 13 C NMR DFT calculation showed good agreement for compound 1, the exomethylene group caused larger deviations in the computed NMR shift values [41,42] of the other derivatives of this work even by testing at different levels of theory.     Long-range heteronuclear NMR correlations between the quaternary carbon C-3 and H-2, H-4, H-5b, and H 3 -15, between C-10 and H-2, H-8, and H 3 -14 signals, and between C-1 and H-2 and H-14 were observed in the HMBC spectrum. The two-and three-bond correlations revealed that the structural fragment together with C-1, C-3, C-10, the C-2 methine, and the 14-methyl group forms a furanoheliangolide skeleton. Detailed analysis of the spectral data suggested that compound 2 is very similar to 8β-isovalerianoyloxy-9α-hydroxycalyculatolide. The difference between the two compounds was the modified α-methylene-γ-lactone moiety. The upfield-shifted chemical shifts of H 2 -13 protons (δ H 3.74 and 3.67) indicated an oxymethylene part in the structure instead of the exomethylene unit. Moreover, the presence of sp 3 methine (δ H -11 2.89, δ C -11 49.3) resonances were observed. These observations led to the conclusion, that the C-11/C-13 double bond was saturated. 1 H-1 H COSY correlations between H-7 and H-11, H-11-and H-13, and longrange correlation between OMe and H 2 -13 proved that a methoxy group is connected at C-13.
The relative configuration of 2 corresponded well with the data reported for furanoheliangolides in the literature, containing an exomethylene moiety at the C-11 position [12]. The NOE correlations between H-8 and H-11 suggested β orientation of the methoxymethylene group at C-11. NOE correlations in a conformationally flexible macrolide ring have to be treated with precaution, since the large flexibility of the ring can lead to wrong assignment as reported in the literature for other related derivatives [43,44]. Thus the relative configuration of 2 was investigated by DFT- 13  The computed 13 (Tables S2 and S3). The DP4+ statistical analysis gave a 99.98% confidence in the gas-phase and 100.00% with SMD solvent model for this isomer [45,46]. The data above corroborate the proposed molecular formula of lobatolide B (2). The ECD spectrum of 2 was recorded both in MeCN and MeOH with different settings but nearly baseline ECD spectra were recorded most likely due to solubility problems. Since +47 value was measured for the specific rotation, this was used instead of the ECD to determine the absolute configuration [47]. CAM-B3LYP/TZVP PCM/CHCl 3 re-optimization of the 136 MMFF conformers resulted in 17 low-energy conformers. OR values were computed for all the conformers at four different levels of theory and they had positive value in all the combinations and the Boltzmann weights were in the range of +53-+71 showing good agreement with the +47 experimental value (Table S4). Thus the absolute configuration of 2 was assigned as (4R,6R,7S,8S,9R,10R,11R) in line with the biosynthetic considerations.
ring. Moreover, this NOE effect is much more probable with the β orientation of H-11 hydrogen than with the α one. The H-8-H-11 interatomic distance in the lowest-energy conformer of (4R,6R,7S,8S,9R,10R,11S)-2 (with α orientation of H-11) is 3.50, while this value is 2.19 Å for the (4R,6R,7S,8S,9R,10R,11R)-2 stereoisomer (with β orientation of H-11) (Figure 4). Thus, the 13 C NMR calculations revealed that the H-8/H-11 NOE correlation unexpectedly derives from the trans relative configuration of the H-8 and H-11 protons. The computed 13 (Tables S2 and S3). The DP4+ statistical analysis gave a 99.98% confidence in the gas-phase and 100.00% with SMD solvent model for this isomer The isovaleroyl group was assigned to C-9 with regard to the HMBC correlations between H-9 and the carbonyl carbon signal (isovaleroyl CO). The only difference between compound 2 and lobatin B is the connection of isovaleroyl (at C-9 in 3, and at C-8 in lobatin B), and hydroxy (at C-8 in 3, and at C-9 in lobatin B) groups.
The relative configuration of compound 3 was studied by means of a NOESY experiment. Cross-peak between H-5 and H 3 -15, and the small coupling constant (J 5 = 1.8 Hz) detected at H-5 proved the Z-configuration of the double bond (C-4-C-5). NOE effects observed between H-7 and H-13b, and H-13b and H-8 dictated the α orientation of these protons. All of the above evidence supported the structure of 3 as 8β-hydroxy-9α-isovaleroyloxyatripliciolide, which was named as lobatolide C.
CAM-B3LYP PCM/MeCN re-optimization of the initial 92 MMFF conformers yielded 13 low-energy conformers, which can be sorted into two groups based on the axial or equatorial arrangement of the C-9 OiVal substituent ( Figure 5). The conformers of a group showed difference in the rotation of the C-9 OiVal group ( Figure 6). The conformers of groups A and B had markedly different computed ECD spectra, while conformers in the same group had similar curves. Although group B conformers had low populations, they had more intense computed ECD spectra than those of group A, and they governed the feature of the sum ECD curve above 240 nm (Figure 7). The Boltzmann-weighted ECD spectra reproduced well the major transitions of the experimental ECD spectrum allowing elucidation of the absolute configuration as (6R,7R,8S,9R,10R), which is in accordance with the biosynthetic considerations.      CHCl3). Based on 1D and 2D NMR data it was determined as lobatin C, isolated previously from N. lobata and N. macrocephala by Passreiter et al. [9,48]. As the compound was identified only by GC-MS analysis [9], and no NMR data were published previously in the literature, detailed NMR studies were performed affording the 1 H and 13 C NMR assignments (Tables 1  and 2). CAM-B3LYP/TZVP PCM/MeCN re-optimization of the initial 109 MMFF conformers of (6R,7S,8S,9R,10R)-4 yielded 10 low-energy conformers and similarly to 3, two conformer groups could be identified with different conformations of the macrolide ring. In group A representing 90.2% sum population, the C-8 OiVal substituent had axial orientation, while it was equatorial in group B conformers (5.6% sum population). The Boltzmann-weighted CAM-B3LYP/TZVP PCM/MeCN ECD spectra reproduced well all the major transitions of the experimental ECD spectrum and hence the absolute configuration was determined as (6R,7S,8S,9R,10R) (Figures 8, S21 and S22). . Based on 1D and 2D NMR data it was determined as lobatin C, isolated previously from N. lobata and N. macrocephala by Passreiter et al. [9,48]. As the compound was identified only by GC-MS analysis [9], and no NMR data were published previously in the literature, detailed NMR studies were performed affording the 1 H and 13 C NMR assignments (Tables 1 and 2). CAM-B3LYP/TZVP PCM/MeCN re-optimization of the initial 109 MMFF conformers of (6R,7S,8S,9R,10R)-4 yielded 10 low-energy conformers and similarly to 3, two conformer groups could be identified with different conformations of the macrolide ring. In group A representing 90.2% sum population, the C-8 OiVal substituent had axial orientation, while it was equatorial in group B conformers (5.6% sum population). The Boltzmann-weighted CAM-B3LYP/TZVP PCM/MeCN ECD spectra reproduced well all the major transitions of the experimental ECD spectrum and hence the absolute configuration was determined as (6R,7S,8S,9R,10R) ( Figure 8, Figures S21 and S22).   . Detailed analysis of spectral data afforded the structure, which was in good agreement with neurolenin B. The missing exomethylene signals (δ H 6.31 and 5.81, br s each), as well as additional sp 3 methine at δ H 3.04 m, δ C 40.2 (C-11) and oxymethylene resonances detected at δ H 3.34 t and 3.68 dd and δ C 66.3 (C-13), indicated the saturation of the C-11/C-13 double bond ( Table 1). The H-7/H-11/H-13 spin system was established by means of 1 H-1 H COSY correlations and HMBC cross peaks of H-7/C-11, H 2 -13/C-7 and H 2 -13/C-11. The 1 H-spectrum contained an additional methoxy signal (δ H 3.39 s), which exhibited HMBC correlation with δ C 66.3 and, therefore, must be situated at C-13. The relative configuration of compound 5, named as lobatolide D, was found to be identical with that of neurolenin B. The presence of trans- The computed 13 C NMR chemical shift data did not show a clear preference for any stereoisomers, and thus the 1 H NMR data were taken also into account (Tables S5-S8). The DP4+ statistical analysis of the combined 13 C and 1 H NMR data gave 99.37% confidence with the SMD solvent model for the (4R,6R,7S,8S,9R,10R,11S)-5 stereoisomer. Interestingly, the (4R,6R,7S,8S,9R,10R,11R) stereoisomer of 5, which is homochiral with 2, gave 0.00% probability at both NMR levels. This result suggested that C-11 chirality center of 2 and 5 was produced by the reduction of the exomethylene moiety, which afforded different C-11 configurations in compounds 2 and 5.
In the knowledge of the relative configuration, the absolute configuration was determined by the TDDFT-ECD protocol performed on the (4R,6R,7S,8S,9R,10R,11S)-5 stereoisomer ( Figure 9). CAM-B3LYP re-optimization of the initial 89 MMFF conformers resulted in 12 low-energy conformers, which gave quite similar ECD spectra ( Figure 10). The Boltzmann-weighted ECD spectra gave a good agreement with the major transitions of the experimental ECD spectrum at all the applied levels of theory allowing elucidation of the absolute configuration as (4R,6R,7S,8S,9R,10R,11S)-5. somer ( Figure 9). CAM-B3LYP re-optimization of the initial 89 MMFF conformers resulted in 12 low-energy conformers, which gave quite similar ECD spectra ( Figure 10). The Boltzmann-weighted ECD spectra gave a good agreement with the major transitions of the experimental ECD spectrum at all the applied levels of theory allowing elucidation of the absolute configuration as (4R,6R,7S,8S,9R,10R,11S)-5.   in 12 low-energy conformers, which gave quite similar ECD spectra ( Figure 10). The Boltzmann-weighted ECD spectra gave a good agreement with the major transitions of the experimental ECD spectrum at all the applied levels of theory allowing elucidation of the absolute configuration as (4R,6R,7S,8S,9R,10R,11S)-5.    (Table 1). Further, the 13 C-JMOD spectrum suggested that the skeleton consists of fifteen carbons ( Table 2) These structural elements, tertiary methyls and quaternary carbons were connected by examination of the long-range C-H correlations detected in the HMBC spectrum. The two-and three-bond correlations between H-3, H-6, H-15, H 2 -2 and the quaternary carbon C-4, and between H 2 -2, H-8, H 2 -9, H-14 and the quaternary C-10 and signals revealed that structural fragment A together with C-10, C-4, and the 14-and 15-methyl groups forms a germacrane skeleton. The lactone ring connected to the macrocycle in position C-6, C-7 was evident from the HMBC cross-peaks between H 2 -13, and C-12, and H 2 -13, H-7 and C-11. The position of the ester groups was proved by the long-range correlation between the ester carbonyl of isovaleroyl group (δ C 173.2) and H-8 (δ H 5.74 d) and the acetyl carbonyl (δ C 171.8) and H-3 (δ H 5.50 d). The remaining epoxy group, which were elucidated from the molecular composition, was placed at C-10-C-1, with regard to the 13 C NMR chemical shifts (δ C-1 65.6 and δ C-10 61.4) and literature data for similar epoxy germacranolides [49]. The relative configuration of the chiral centers was studied by NOESY measurements. Diagnostic NOE correlations were detected between H-6 and All of the above evidence was used to propose the structure of this compound as depicted in structural formula 6. Compound 6 is the 8-epimer of 8α-isovaleryloxy-8-desacylviguestenin, reported earlier from Viguiera procumbens (Asteraceae) [49] Moreover, in contrast to 8α-isovaleryloxy-8desacylviguestenin, the trans position of double bond between C-4-C-5 was proved by the NOE correlations between H-6 and H 3 -15 (above the plane of the macrocycle) and between H-3 and H-5 (below the plane of the ring). Therefore, lobatolide E was elucidated as depicted structural formula 6.
ECD calculations performed for the 10 low-energy CAM-B3LYP conformers of (1R,3S, 6R,7R,8R,10R)-6 obtained from the re-optimization of the initial 74 MMFF conformers gave nice overall agreement with the experimental ECD spectrum (Figure 11). In all the 10 computed conformers, the C-8 OiVal substituent adopted axial orientation and the conformers gave very similar ECD spectra allowing elucidation of the absolute configuration of 6 as (1R,3S,6R,7R,8R,10R) ( Figure 12).    Comparison of the NMR data of part A with those of sesquiterpenes previously isolated from the plant showed great similarities of unit A with neurolenin B. However, the absence of exomethylene and H-7 signals, as well as the appearance of proton resonances at δ H 3.05 (dd, J = 14.5, 8.8 Hz) and δ H 2.48 (dd, J = 14.5, 6.8) attributed to a saturated methylene suggested the relocation of the double bond from C-11-C-13 to C-7-C-11. This conclusion was supported by the significantly downfield-shifted signal of H-8 (δ H 6.38) (neurolenin B: δ H 5.56), in addition with HMBC correlations observed between the quaternary olefinic C-7 (δ C 154.2) and H-6, H-8 and H-13a. Careful interpretation of the spectral data allowed determining part B as a 8β-isovaleroyloxy-9α-hydroxycalyculatolide unit. Chemical shifts of H-5 (δ H 3.54 m, 1H) and C-5 (δ C 45.6) clearly indicated that part B is connected to A in position C-5. In addition, with the 1 H- 1  ). In addition, the 13 C and JMOD spectra suggested a sesquiterpene core consisting of one methyl, five methylenes, five methines and four quaternary carbons (Table 3). Two structural elements could be assigned based on the 1 H-1 H COSY spectrum: −CHR-CH 2 -CH 2 − (A δ H 3.50, 1.81, 1.58 [50]. All of the above evidence proved the eudesmanolide structure 8β-isovaleroyloxyreynosin for this compound, and named as lobatolide G. ECD calculations were performed for (1R,5S,6R,7R,8R,10R)-8 similarly to the above compounds, but only partial agreements were found, and the experimental spectrum was weak and noisy even after measuring in various solvents and concentrations/settings. Therefore, the absolute configuration was verified by computing for the closely related derivative 9 (see below).
Naturally occurring germacranolides arise from all-trans farnesyl pyrophosphate by a series of biochemical transformations. Compounds belonging to this group (e.g., costunolide) primarily contain two endocyclic double bonds with all-trans configuration [e.g., costunolide derivatives]. However, in many cases cis configuration also occurs, e.g., in lobatolide C (3). The stereochemical skeletal types have distinct conformations, even allowing a certain amount of conformational flexibility [54]. In many cases, one of the endocyclic double bonds has undergone epoxidation as can be found in lobatolide E (6) or give a furan-type germacranolide occur in calyculatolide derivatives, like compound 7 (unit B), and 1-4. was first identified from the fruits of Aphanamixis grandifolia [52,53].
Since the absolute configuration of volenol (9) was not described in the literature, th above TDDFT-ECD protocol was applied to (1R,5S,6S,7S,10R)-9 ( Figure 13). CAM-B3LYP re-optimization of the initial 24 MMFF conformers resulted in 12 low-energy conformers the ECD spectra of which resembled well the experimental ECD spectrum (Figure 14) Therefore, the absolute configuration of 9 was verified in accordance with the biosyntheti considerations as (1R,5S,6S,7S,10R).  Naturally occurring germacranolides arise from all-trans farnesyl pyrophosphate by a series of biochemical transformations. Compounds belonging to this group (e.g costunolide) primarily contain two endocyclic double bonds with all-trans configuration [e.g., costunolide derivatives]. However, in many cases cis configuration also occurs, e.g in lobatolide C (3). The stereochemical skeletal types have distinct conformations, even Since the absolute configuration of volenol (9) was not described in the literature, the above TDDFT-ECD protocol was applied to (1R,5S,6S,7S,10R)-9 ( Figure 13). CAM-B3LYP re-optimization of the initial 24 MMFF conformers resulted in 12 low-energy conformers, the ECD spectra of which resembled well the experimental ECD spectrum ( Figure 14). Therefore, the absolute configuration of 9 was verified in accordance with the biosynthetic considerations as (1R,5S,6S,7S,10R).  Naturally occurring germacranolides arise from all-trans farnesyl pyrophosphate by a series of biochemical transformations. Compounds belonging to this group (e.g., costunolide) primarily contain two endocyclic double bonds with all-trans configuration [e.g., costunolide derivatives]. However, in many cases cis configuration also occurs, e.g., in lobatolide C (3). The stereochemical skeletal types have distinct conformations, even

Antiproliferative Activity of Isolated Compounds In Vitro
The antiproliferative activities of the isolated compounds 1-7, 9, 10 and neurolenin B (unit A of compound 7) were tested on three cervical cancer cell lines of different human papilloma virus (HPV) of different status (SiHa, HeLa, and C33A). Many of the compounds were additionally tested on non-cancerous cell lines NIH/3T3 (mouse embryonic fibroblast) and MRC-5 (human fibroblast) to obtain information concerning the cancer selectivity of the isolated natural products (Table 4). Compound 4 and neurolenin B exhibited higher potency than clinically utilized reference agent cisplatin while compounds 1, 3, 6 and 7 exerted similar antiproliferative properties to those of cisplatin. None of the presented natural products displayed substantial cancer selectivity exception for 6 which resulted in IC 50 values in the range of 3.2-3.6 µM on C33A and SiHa cells but above 11 µM on the two fibroblast cell lines. Neurolenin B was proved to be the most active molecule with IC 50 values against cancer cells lower than 1.4 µM on all cancer cell lines and NIH/3T3 while its calculated IC 50 was slightly higher against human fibroblasts, indicating that the cancer selectivity is limited. Based on Pharmaceutics 2021, 13, 2088 20 of 25 these antiproliferative activities 6 was selected for additional experiments. HeLa was shown to be the least sensitive cell line from the three cervical cancers. Since HeLa and SiHa cells result from HPV-mediated transformation, HPV-positivity does not explain the different responses of the two cell lines. The most invasive SiHa cell line was chosen further to investigate the possible mechanisms behind the antiproliferative effects. SiHa cells carry HPV-16 genes that contribute to the formation of metastases of this cancer type. HPV-16 oncoproteins promote cervical cancer invasiveness by upregulating specific matrix metalloproteinases (MMPs). HPV-positive cell lines express higher levels of MMP-2, membrane type 1-MMP and tissue inhibitor of metalloproteinase 2 [55]. Based on the antiproliferative assays, cell cycle analysis was performed on SiHa cells. Compound 6 caused a significant concentration-dependent accumulation of cells in the S phase to the detriment of G0/G1 phase after 48 or 72 h treatment ( Figure 15). Cell cycle arrest in the S phase might refer to the cells having a higher tendency to enter S phase due to the dysfunction of cyclin dependent kinases, or it can be a consequence of inhibited DNA synthesis. The molecular mechanism of this cell cycle blockade requires further elucidation. Iso-seco-tanapartholide 3-O-methyl ether elicited similar S phase accumulation in HL-60 leukemia cells after 24 h of incubation at low concentration (5 µM). This phenomenon was accompanied by G2/M cell cycle phase arrest and subG1 accumulation, which could not be observed in the cell cycle profile of compound 6 [56]. G2/M phase accumulation appears to be a more characteristic effect in cervical cell lines treated by SLs. ROS generation, caspase-3 activation, inhibition of Bcl-2, and enhancement of Bax protein transcription seems a common background of in vitro anticancer activities [57,58]. A sesquiterpene lactone component of Elephantopus mollis (EM23) caused cell accumulation in the S phase parallel to the G2/M growth in K562 and HL-60 leukemia cells. ROS generation seems a grounded explanation for the pro-apoptotic effects since ROS quenching with N-acetyl-L-cysteine almost completely reversed these observations [59]. Compound 6 with high antiproliferative potency and modest selectivity to cancerous cells were chosen in order to assess its antimetastatic activity. The migration of cancer cells is an important property to form metastases. Therapeutical modalities that target the dissemination of tumor cells from the primary tumor could significantly improve the success of cancer therapy. Wound healing assay represents a simple tool to follow the antimigratory effects of SLs. Wound healing assay was performed on SiHa cell line with compound 6 at 1.5 µ M and 3 µ M concentrations. The inhibition of wound healing was evaluated by the measurement of the wound surface. 30% wound closure was detected after 24 h in the Compound 6 with high antiproliferative potency and modest selectivity to cancerous cells were chosen in order to assess its antimetastatic activity. The migration of cancer cells is an important property to form metastases. Therapeutical modalities that target the dissemination of tumor cells from the primary tumor could significantly improve the success of cancer therapy. Wound healing assay represents a simple tool to follow the antimigratory effects of SLs. Wound healing assay was performed on SiHa cell line with compound 6 at 1.5 µM and 3 µM concentrations. The inhibition of wound healing was evaluated by the measurement of the wound surface. 30% wound closure was detected after 24 h in the control wells, while 3 µM treatment with compound 6 resulted in the inhibition of wound healing by causing only 12% closure. After 48 h, the controls showed 70% wound closure; however, the 1.5 and 3 µM treated cells migrated only to 47% and 18% of the wound surface, respectively ( Figure 16). These results follow the literature on the effects of sesquiterpenes. Parthenolide and other SLs inhibit cell migration by suppressing the FAK1 signaling pathway in breast cancer cultures [60]. Artesunate, an artemisinin derivative, promotes the downregulation of COX-2 expression associated with lymph node metastasis in cervical cancers. Conversely, COX-2 overexpression and consequent PGE2 formation can be responsible for the enhanced expression of metalloproteinases and vascular endothelial growth factors [61].
In the antiproliferative assay, lobatolides C (3), and E (6), neurolenin B (unit A of 7), and lobatolide F (7) were proved to be the most active compounds against HeLa, C33A, and SiHa cells. Although, neurolenin B showed the lowest IC 50 values against cancer cells, it also inhibited normal cells at low concentration. Considering the selectivity of the compounds, lobatolide E (6) seems to be the most promising one. Moreover, this compound caused a significant, concentration-dependent accumulation of SiHa cells in the S phase during the cell cycle analysis investigation. The antimetastatic activity of compound 6 was also confirmed by wound healing assay.