Sesquiterpene Lactones from Artemisia absinthium. Biotransformation and Rearrangement of the Insect Antifeedant 3α-hydroxypelenolide

Three new compounds, the sesquiterpenes absilactone and hansonlactone and the acetophenone derivative ajenjol, have been isolated from a cultivated variety of Artemisia absinthium. In addition, the major lactone isolated, 3α-hydroxypelenolide, was biotransformed by the fungus Mucor plumbeus affording the corresponding 1β, 10α-epoxide. A cadinane derivative was formed by an acid rearrangement produced in the culture medium, but not by the enzymatic system of the fungus. Furthermore, 3α-hydroxypelenolide showed strong antifeedant effects against Leptinotarsa decemlineata and cytotoxic activity to Sf9 insect cells, while the biotransformed compounds showed antifeedant postingestive effects against Spodoptera littoralis.


Introduction
The Artemisia genus with more of 400 species is one of the largest genera in the Asteraceae family. The species Artemisia absinthium L., known as wormwood, is a perennial medicinal species which has been widely investigated due to its ethno-pharmacological interest.
A. absinthium is abundant in the mountains of Spain as a ruderal species. There are seven chemotypes described in the Iberian Peninsula [1]. A thujone-free population of this plant (A. absinthium var. candial) has been domesticated for the production of essential oil [2]. The oil characterized by the presence of cis-epoxyocimene, (−)-cis-chrysanthenol, chrysanthenyl acetate, linalool and trans-caryophyllene showed strong antifungal effects [2]. Furthermore, two ocimene monoterpenes were isolated and their absolute configurations determined by vibrational circular dichroism (VCD) [3].
However, the non-volatile constituents of A. absinthium have received little attention. This plant is characterized by its content in the sesquiterpene lactones hydroxypelenolide (3) and ketopelenolides A and B (4)(5) [4][5][6]. Additionally, five sesquiterpene lactones, a flavone [7] and a germacrane lactone artabolide with auxin transport inhibitor properties [8] have been also reported. A previous study on the domesticated A. absinthium var. candial reported the lactone hydroxypelenolide (3), with insect antifeedant effects, and the flavones artemetin and casticin [6] as the main components of its non-volatile extract [6].
Biotransformations of terpenoids can modify the biological activities of the starting compounds [9] and are also used to study structure-activity relationships of bioactive compounds [10]. In this context, Mucor plumbeus, with a broad substrate specificity [11,12], has been used in biotransformations of diterpenes to develop models to explain their hydroxylations [13][14][15][16][17] and for the biotransformation of an africanane sesquiterpene to give epoxy derivatives with improved insect antifeedants and insecticidal effects [18]. In this work we describe three unknown compounds, the sesquiterpene lactones absilactone (1) and hansonlactone (2) and the acetophenone derivative artenol (12) [4], isolated from A. absinthium var. candial along with several known compounds. Furthermore, 3α-hydroxypelenolide (3), previously known as hydroxypelenolide [4], was biotransformed by Mucor plumbeus to give 1β, 10α-epoxide (9) and a cadinane derivative (10). The latter was formed by an acid rearrangement produced in the culture medium, not by the enzymatic system of the fungus. We also describe the insecticidal activities of these compounds against the insect pests Spodoptera littoralis, Myzus persicae, the cytotoxic effects on Spodoptera frugiperda pupal ovarian tissue cells (Sf9), and the phytotoxic effects on Lactuca sativa.

Components of A. absinthium
Absilactone is a new nor-sesquiterpene, which structure has been determined as 1 based on the following considerations: Its high-resolution mass spectrum showed the molecular ion at m/z 248.1040, which corresponds to the molecular formula C 14 H 16 O 4 . In the 1 H-NMR spectrum three methyl groups were observed, two of which are over double bonds, resonating as singlets at δ H 2.23 and 2.36, while the third corresponds to a secondary methyl that appears as a doublet at δ H 1.25 (J = 7.0 Hz). The geminal proton (H-6) at the lactonic ring closure resonates at δ H 4.85. Its coupling constant with H-7 (10.6 Hz), indicated a trans-relationship between these H-6 and H-7 hydrogens ( Figure S1).
The 13 C NMR spectrum showed, in addition to the three methyl groups, two methylenes at δ C 26.0 and 37.6, the relatively low value of the first is typical of being located between another methylene and a methine, so it was assigned to C-8. The methine group at C-11 was observed at δ C 41.5. Other signals present in this spectrum were six singlets, two of them at δ C 166.8 and 177.6 characteristic of the carbonyl groups of the two lactones, while the other four singlets correspond to carbons of the two tetrasubstituted double bonds ( Figure S2).
Austroyunnane F (7) is a nor-sesquiterpene of the absilactone type, which was isolated from A. austro-yunnanensis [19]. This compound and absilactone (1) probably derive from a similar biosynthetic pathway, considering that both possess a similar framework and have been obtained from species of the Artemisia genus. Another analogous nor-sesquiterpene but with a pseudoguaiane skeleton, 4-hydroxy-nor-psilotropin (8), has been obtained from Psilotrophe villosa [20].
Another undescribed compound isolated from this plant was an acetophenone derivative, which we have named ajenjol. Its structure 12 was assigned considering its spectroscopic data: In the HRMS the molecular ion appears at m/z 250.1201, which corresponds to the molecular formula C14H18O4. The 1 H NMR spectrum showed resonance of the two aromatic protons as singlets at δH 6.45 and 8.25, the former located between carbons bearing oxygens and the latter situated in ortho position to the acetyl group. Signals of four methyl groups were observed, two from the side chain at δH 0.95, one from the acetyl group at δH 2.62 and another from the methoxy group at δH 3.94. A doublet of the two H-10, located on a carbon in α-position to a carbonyl group, appears at δH 2.81, and the H-11 methine resonates as a double triplet at δH 2.21. A singlet at δH 12.92 was due to the hydroxylic hydrogen bonded with the carbonyl of the acetyl group ( Figure S13).  Figure S14). In the HMBC spectrum, the following connectivities were observed: H-3 with C-1/C-2/C-5; H-6 with C-2/C-4; H-8 with C-7; H-11 with C-9/C-12/C-13; Another undescribed compound isolated from this plant was an acetophenone derivative, which we have named ajenjol. Its structure 12 was assigned considering its spectroscopic data: In the HRMS the molecular ion appears at m/z 250.1201, which corresponds to the molecular formula C 14 H 18 O 4 . The 1 H NMR spectrum showed resonance of the two aromatic protons as singlets at δ H 6.45 and 8.25, the former located between carbons bearing oxygens and the latter situated in ortho position to the acetyl group. Signals of four methyl groups were observed, two from the side chain at δ H 0.95, one from the acetyl group at δ H 2.62 and another from the methoxy group at δ H 3.94. A doublet of the two H-10, located on a carbon in α-position to a carbonyl group, appears at δ H 2.81, and the H-11 methine resonates as a double triplet at δ H 2.21. A singlet at δ H 12.92 was due to the hydroxylic hydrogen bonded with the carbonyl of the acetyl group ( Figure S13).
The 13 C-NMR spectrum confirmed the presence of the aromatic ring, with resonances of two doublets at δ C 99.8 and 135.2, located between carbons bearing oxygens and carbonyl groups, respectively, and four singlets to δ C 113.8, 121.0, 164.8 and 167.5. The last two carbons linked to methoxy and hydroxy groups, respectively. The two carbonyl groups appeared in this spectrum at δ C 199.5 and 203.4, the latter corresponding to the acetyl group ( Figure S14). In the HMBC spectrum, the following connectivities were observed: H-3 with C-1/C-2/C-5; H-6 with C-2/C-4; H-8 with C-7; H-11 with C-9/C-12/C-13; H-12/H-13 with C-10; -OH with C-1 and H-14 (-OMe) with C-4. In the NOESY experiment, Ajenjol was assigned to structure 12 on the basis of the following considerations: We have now described and assigned the 13 C NMR spectrum ( Figure S12) of espeletone (11) [21], which values of C-10. C-11, C-12 and C-13 in its side chain and those of ajenjol (12) were identical (Table 1). This fact indicated that both compounds have a methoxy group at the C-4 position, which was confirmed observing that the resonance values above indicated for 11 and 12 were different for the corresponding carbons in 14. This last product, isolated from Polymnia sonchifolia [22], has a hydroxyl group at C-4 with the hydrogen bonded to the C-9 carbonyl, affecting the carbon resonances of the side chain, especially to C-10. The reported 13 C NMR data of 14 have been assigned here (Table 1). Structure 12 of ajenjol, and not 13, was also in accordance with the chemical shift of the C-7 carbonyl. Thus, this carbon appears in 12 at δ C 203.4, a value more similar to that of 6-hydroxytremetone (15) (δ C 201.9) [23] than that of 6-methoxytremetone (16) (δ C 197.9) [24]. On the other hand, espeletone (11), which we have now also isolated from A. absinthium, could be a possible biogenetic precursor of this new compound, ajenjol (12). Structure 17, with an OH group at C-4, had been assigned to glutinosol, which was obtained from A. glutinosa [25]. Unfortunately, the 13 C NMR data for this compound were not reported, and the 1 H NMR spectrum described can be valid for 17 but also for 18. Indeed, the presence of espeletone (11) in this plant, and its 10, 11-dehydro derivative, indicates that 18 could be an alternative structure.

Biotransformation and Rearrangement of 3α-hydroxypelenolide (3)
During the past years we have been interested in the microbiological transformation of diterpenes by the fungus M. plumbeus [13][14][15][16][17]. The aim of these studies has been to develop models to explain the hydroxylation of these compounds by this microorganism, which possesses a broad specificity in the substrate [11,12]. To complement these works, in order also to study their structure-activity relationship as potential pesticides, we expand this research to the sesquiterpenes. Thus, we have investigated the biotransformation of an africanane sesquiterpene by this fungus [18]. Now, continuing with these studies, and considering the good yield of 3α-hydroxypelenolide isolated from A. absinthium, we have biotransformed it by the fungus M. plumbeus affording two products 9 and 10.
The less polar of the compound isolated was 9, which showed a 1 H NMR spectrum with very broad signals and a 13 C NMR with few resonances. These effects are due to the conformational flexibility that possesses the 10-membered ring of some germacranolides at ambient temperature (298 • K). To obtain these spectra in better conditions we ran them at 233 • K, although in fact at this temperature very small couplings could not be observed. Their NMR spectra, in comparison with that of substrate ( Figures S5 and S6), showed lack of the double bond resonances, which were substituted by the corresponding to an oxirane ring at δ H 3.28 (H-1) and δ C 58.7 (C-1) and 60.3 (C-10) (Figures S7 and S8). Thus, in the HMBC experiment, connectivities were observed of H-1 with C-2/C-3; H-2 with C-1/C-10; H-9 with C-10; and H-14 with C-1/C-10. The relative low field resonance at δ H 3.28 of H-1, the geminal proton to the epoxy group, was due to the presence of the 3α-OH, which also permitted H-1 to be assigned an α-stereochemistry. The geminal methyl (C-14) to the epoxide at C-10 was given a β-stereochemistry, because it showed a crosspeak with H-6 in the NOESY experiment. Thus, the epoxide ring has a trans-geometry as also had the double bond of the substrate. The chair-chair conformation of the 10-membered ring at 233 • K was determined also considering NOESY data. Thus, correlations of H-6 with H-4/H-8β/H-14 indicated that they have an axial configuration in the β-face of the molecule, whilst correlations of H-1 with H-7/H-9α showed that these hydrogens were located in the α-face. This molecular conformation was confirmed by computational analysis, and resulted similar to that described for another 1β, 10α-epoxy-11, 13-dihydrogermacranolide, isolated from Achillea crithmifolia [36,37].
Compound 10, also obtained in the fermentation of the substrate 3, was not formed by the enzymatic system of M. plumbeus, but by acid rearrangement of 3 in the culture medium ( Figure 1). This fact was confirmed in another experiment carried out under the same conditions but in the absence of the fungus, where the incubation of compound 3 did not afford the epoxide 9. The structure 10 assigned to this product was based in the following considerations: Its HRMS showed the fragment of higher mass at m/z 255.1596 (C 14 H 23 O 4 ), which is formed from the molecular ion by loss of a methyl group. Thus, its molecular formula was C 15 H 26 O 4 . In the NMR spectra does not appear the resonances of the double bond and the geminal proton to the lactone closure, which have taken part in the rearrangement, being substituted by a methylene group at δ H 1.22 and 2.10 (δ C 34.2, C-2), a methine at δ H 1.29 (δ C 42.1, C-6), and a tetrasubstituted carbon bearing a hydroxy group at δ C 72.6 (C-10). The carbon resonance of the acid group, formed by opening of the lactone, appears at δ C 179.3 ( Figures S9 and S10). The corresponding HMBC connectivities were H-2 with C-1/ C-3/C-4/C-6; H-11 with C-6/C-7/C-8/C-13; H-13 with C-7/C-11/C-12 and H-14 with C-1/C-9/C-10. The H-1/H-6 trans-relationships was determined considering the NMR signal of H-1 (δ H 1.53, td, J = 10.8, 3.1 Hz), which indicated diaxial interactions of this hydrogen with H-6 and H-2β, and an axial-equatorial coupling with H-2α. The configuration at C-10 was resolved take into consideration the resonance of the C-14 methyl at δ C 21.0, which is typical of an axial stereochemistry, because the chemical shifts described for axial and equatorial orientation of this methyl are δ C 21.6 and 28.0, respectively [38]. This stereochemistry was confirmed in the NOESY spectrum with an axial correlation of H-2β with H-14.
HMBC experiment, connectivities were observed of H-1 with C-2/C-3; H-2 with C-1/C-10; H-9 with C-10; and H-14 with C-1/C-10. The relative low field resonance at δH 3.28 of H-1, the geminal proton to the epoxy group, was due to the presence of the 3α-OH, which also permitted H-1 to be assigned an α-stereochemistry. The geminal methyl (C-14) to the epoxide at C-10 was given a β-stereochemistry, because it showed a crosspeak with H-6 in the NOESY experiment. Thus, the epoxide ring has a trans-geometry as also had the double bond of the substrate. The chair-chair conformation of the 10-membered ring at 233 °K was determined also considering NOESY data. Thus, correlations of H-6 with H-4/H-8β/H-14 indicated that they have an axial configuration in the β-face of the molecule, whilst correlations of H-1 with H-7/H-9α showed that these hydrogens were located in the α-face. This molecular conformation was confirmed by computational analysis, and resulted similar to that described for another 1β, 10α-epoxy-11, 13-dihydrogermacranolide, isolated from Achillea crithmifolia [36,37].
Compound 10, also obtained in the fermentation of the substrate 3, was not formed by the enzymatic system of M. plumbeus, but by acid rearrangement of 3 in the culture medium ( Figure 1). This fact was confirmed in another experiment carried out under the same conditions but in the absence of the fungus, where the incubation of compound 3 did not afford the epoxide 9. The structure 10 assigned to this product was based in the following considerations: Its HRMS showed the fragment of higher mass at m/z 255.1596 (C14H23O4), which is formed from the molecular ion by loss of a methyl group. Thus, its molecular formula was C15H26O4. In the NMR spectra does not appear the resonances of the double bond and the geminal proton to the lactone closure, which have taken part in the rearrangement, being substituted by a methylene group at δH 1.22 and 2.10 (δC 34.2, C-2), a methine at δH 1.29 (δC 42.1, C-6), and a tetrasubstituted carbon bearing a hydroxy group at δC 72.6 (C-10). The carbon resonance of the acid group, formed by opening of the lactone, appears at δC 179.3 ( Figures S9 and S10). The corresponding HMBC connectivities were H-2 with C-1/ C-3/C-4/C-6; H-11 with C-6/C-7/C-8/C-13; H-13 with C-7/C-11/C-12 and H-14 with C-1/C-9/C-10. The H-1/H-6 trans-relationships was determined considering the NMR signal of H-1 (δH 1.53, td, J = 10.8, 3.1 Hz), which indicated diaxial interactions of this hydrogen with H-6 and H-2β, and an axial-equatorial coupling with H-2α. The configuration at C-10 was resolved take into consideration the resonance of the C-14 methyl at δC 21.0, which is typical of an axial stereochemistry, because the chemical shifts described for axial and equatorial orientation of this methyl are δC 21.6 and 28.0, respectively [38]. This stereochemistry was confirmed in the NOESY spectrum with an axial correlation of H-2β with H-14.  Cadinane diterpenes of this type had been isolated from the plant Leucanthemopsis pulverulenta, which also contains germacranolides of heliangolide type [39,40]. Later, it was suggested that the rearrangement of these lactones could led to the formation of the cadinane derivatives, which was confirmed making the same transformation by treatment with BF 3 Et 2 O [41]. Now, regarding the rearrangement described in this work, we must highlight the very mild conditions and the good specificity with which it has taken place.

Biological Activity of 3α-hydroxypelenolide and Biotransformed Products 9 and 10
The insecticidal, antifeedant and phytotoxic effects of compound 3 and compounds obtained by its biotransformation (9 and 10), were studied.
When orally injected to Spodoptera littoralis larvae, a moderate antifeedant postingestive effect was observed for 9 that increased for compound 10, with values of 79 and 81% larval weight gain (∆B) and consumption (∆I) respect to the control, respectively, without additional toxic effects (pANCOVA2 > 0.05) The cytotoxic effects of compound 3 on the insect cells Sf9 disappeared for the two compounds obtained by biotransformation (9 and 10) ( Table 2). Sesquiterpene lactones present a wide range of biological activities, including cytotoxic, antitumoral, antimicrobial, insecticidal and phytotoxic [42]. In our study, the insect antifeedant cytotoxic effects of 3 decreased when the double bond between the C-1 and C-10 positions disappeared through a process of oxidation that ended with the formation of an epoxide (9). However, analogous of 9, ivaxillin and eriolin isolated from Carpesium abrotanoide, have shown antifeedant effects to lepidopteran larvae [43].
In tests carried out with germacranolides such as costunolide, parthenolide, 1, 10epoxicostunolide among others, on L. sativa seeds, it was observed that introduction of an epoxide in the molecule produced a decrease of the germination activity and an increase in radicle length. Additionally, the absence of exocyclic methylene in C-13, transformed the activity on radicle length into inhibitory activity [44]. These data agree with the results obtained in our case, since the introduction of the epoxy (9) eliminated the low inhibitory activity on radicle of compound 3.
Cadinane (10), with eudesmanolide structure, was the only one that showed postingestive effects. Compound 10 is an analog of artemisinin acid, a compound isolated from A. annua and used for the artemisinin semi-synthesis. Cadinane-type sesquiterpenes showed insecticidal and ixodicidal effects [45]. An analogous of 10, cadine-4,10(15)-dien-3-one, produced toxicity in adults of Cylas formicarus and sterility in the tick Boophilus microplus [46]. According to Buchanan et al. [45], the C-3 and C-4 sterochemistry of cadine-4,10 (15)-dien-3one derivatives, have an important effect on the insecticidal properties of these compounds, decreasing the activity when the C-4 configuration is R and when the stereochemistry of the 3α-hydroxyl is reversed. Cadinane-type sesquiterpenes also showed antigermination and phytotoxic activity against lettuce and radish seeds [45,47,48]. However, 10 was not phytotoxic in our experiments.

General Experimental Procedures
Melting points were determined with a Reichert Thermovar apparatus and are uncorrected (Reichert Technologies, Buffalo, NY, USA). Optical rotations were determined at room temperature on a Perkin Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA). IR spectra were taken in a Bruker IFS 66/S spectrometer. NMR spectra were run on a Bruker AMX-500 spectrometer with pulsed field gradient using the solvent (CDCl 3 ) as internal standard (Bruker Corporation, Billerica, MA, USA). EIMS and exact mass mea- surements were recorded on a Micromass Autospec instrument at 70 eV. Preparative and semipreparative HPLC was carried out with a Beckman Coulter 125P equipped with a diode-array detector Beckman Coulter 168 (Beckman Coulter Life Sciences, Brea, CA, USA) and preparative Interstil Prep-sil 20 mm × 250 mm, 10 µm particle size (Gasukuro Kogio, Shinjuku-ku, Tokyo, Japan) and semipreparative Ultrasphere silica 10 mm × 250 mm, 5 µm particle (Beckman Coulter Life Sciences, Brea, CA, USA) size columns. Silica gel 60 F 254 (Merck 105715, Darmstadt, Germany) and Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA) were used for column chromatography. Computational analysis was carried out with the Hyperchem 7.0 program applying the Polak-Ribiere minimization algorithm.

Plant Material
Artemisia absinthium L. was cultivated in an experimental field at Ejea de los Caballeros, Zaragoza, Spain, at 345 m of altitude, with exemplars obtained from natural populations at San Blas (Teruel, Spain). A detailed description of the cultivar has been described [49]. Vegetal material was collected in flowering state, which was subjected to a drying process in the shade, with air flow, for eight days.

Extraction and Isolation
Dry plant (3.5 kg) was left in maceration with acetone at room temperature for four days. The cold extract was filtered and concentrated in vacuo to afford a syrup gum (246 g).

Cytotoxicity
Sf9 cells derived from Spodoptera frugiperda pupal ovarian tissue (European Collection of Cell Cultures, ECCC) were maintained in TC-100 insect cell medium supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin/streptomycin at 26 • C. Cells seeded in 96-well flat-bottom microplates with 100 µL medium per well, were exposed for 48 h to serial dilutions of the test compounds in DMSO (<1% final concentration). Cell viability was analyzed by the MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) colorimetric assay method and the purple-colored formazan precipitate was dissolved with 100 µL of DMSO as described by González-Coloma et al. [53]. The active compounds were tested in a dose-response experiment to calculate their relative potency (IC50) values, the effective dose to give 50% cell viability, which was determined from linear regression analysis (% cell viability on log dose).

Phytotoxicity
These experiments were conducted with Lactuca sativa var. Carrascoy seeds as described by [54]. The germination was monitored daily for 6 days and the radicle length measured at the end of the experiment (20 roots randomly selected for each experiment digitalized with https://imagej.nih.gov/ij//, accessed on 20 April 2021) [52]. A nonparametric analysis of variance (ANOVA) was performed on germination and radicle length data (STATGRAPHICS Centurion XVI, version 16.1.02).
The major compound 3, 3α-hydroxypelenolide, was a strong antifeedant against Leptinotarsa decemlineata and moderate antifeedant against Myzus persicae. Its biotransformation products, 9 and 10, were not antifeedant. None of these compounds were phytotoxic against Lactuca sativa. When orally injected to Spodoptera littoralis larvae, a moderate antifeedant postingestive effect was observed for 9 that increased for compound 10. The cytotoxic effects of compound 3 on the insect cells Sf9 disappeared for the two compounds obtained by biotransformation (9 and 10).