New 11-Methoxymethylgermacranolides from the Whole Plant of Carpesium divaricatum

Eight new 11-methoxymethylgermacranolides (1–8) were isolated from the ethanol extract of the whole plant of Carpesium divaricatum. The planar structures and relative configurations of the new compounds were determined by detailed spectroscopic analysis. The absolute configuration of 1 was established by electronic circular dichroism (ECD) spectrum and X-ray crystallographic analysis, and the stereochemistry of the new compounds 2–8 were determined by similar ECD data with 1. The absolute configurations of 5 and 7 were further confirmed by using quantum chemical electronic circular dichroism (ECD) calculations. Compound 4 exhibited weak cytotoxicity against MCF-7 cells. Compound 8 could potently decrease PGE2 productions in LPS-induced RAW 264.7 cells.


Introduction
Sesquiterpenoid lactones have in many instances been instrumental in providing interesting leads for drug development against numerous diseases [1][2][3]. Among many other examples, the class of germacranolides has attracted a great deal of attention in recent years. Parthenolide, a germacranolide isolated from Tanacetum parthenium, exhibited promising antitumor efficacy [3,4]. Germacranolides are one class of the main sesquiterpene lactones, reported with broad bioactivities, including cytotoxicity, anti-inflammation, and antimalarial action [4][5][6][7]. In the past five years, germacranolides have been reported in more than 250 publications [4]. These germacranolides contain a plethora of stereogenic centers and a multitude of oxygenated functionalities, creating the problem of the assignment of absolute configuration.
In our ongoing search for new/novel and bioactive products from the medicinal plants in China, Carpesium divaricatum Sieb.et Zucc, belonging to the family Compositae, were found to be rich in highly oxygenated germacranolides [6][7][8][9][10]. Our previous study led to the distinction between four subtypes of these germacranolides [11][12][13][14]. A further investigation of C. divaricatum was conducted, resulting in the isolation of eight new 11methoxymethylgermacranolides (1-8). Notably, compounds 1-8 represent a new subtype V (named 3-oxo-11-methoxymethylgermacranolide), possessing a 6,7-γ-lactone ring and the 3-ketone group. Subtypes IV (the basic structure of cardivarolides) and V have similar skeletons except for the presence of a methoxymethyl group instead of the ∆ 11,13 exocyclic methylene group in the five-membered ring [12,13] (Figure 1). In this paper, the isolation, structural elucidation, absolute configuration, and bioactive evaluation of these compounds are presented.
Compounds 5-6 shared the same molecular formula C 26 H 42 O 10 , established from their HRESIMS ions at m/z 537.2686 [M + Na] + and m/z 537.2688 [M + Na] + . The 1 H and 13 C NMR data of 5-6 showed great similarity with those of 1, except for the ester residues at C-5 and C-9. An isobutyryloxy group at C-5 and an angeloyloxy group at C-9 in 1 were placed by two 3-methylbutyryloxy groups of 5 and a 2-methylbutyryloxy group and a 3-methylbutyryloxy group of 6, respectively. These observations were confirmed by analyses of relevant 1 H− 1 H COSY, HSQC, and HMBC data. Similarly, their relative configurations were determined as the same as that of 1 by comparison of the ROESY data. The absolute configuration of 5 was established by using quantum chemical electronic circular dichroism (ECD) calculations. Due to the huge amounts of conformations from its numerous single bonds, a simplified structure named 5Ja (Supporting Information C1), in which two acetyl groups instead of the 3-methylbutyryloxy moieties, was used for ECD calculations [16]. The calculated ECD spectrum ( Figure 5) of (4R, 5R, 6S, 7R, 8R, 9R, 10R, 11R)-5Ja agreed well with the experimental spectrum and confirmed the (4R, 5R, 6S, 7R, 8R, 9R, 10R, 11R) absolute configuration. Based on biosynthetic considerations [4,13], similar ECD data of 6 and 5 revealed the same absolute configuration of 6 as that of 5. Thus, the structures of compounds 5-6 were established, as shown in Figure 2, named 11-methoxymethylcardivarolide K and 11-methoxymethylcardivarolide L, respectively. configuration of 5 was established by using quantum chemical electronic circular dichroism (ECD) calculations. Due to the huge amounts of conformations from its numerous single bonds, a simplified structure named 5Ja (Supporting Information C1), in which two acetyl groups instead of the 3-methylbutyryloxy moieties, was used for ECD calculations [16]. The calculated ECD spectrum ( Figure 5) of (4R, 5R, 6S, 7R, 8R, 9R, 10R, 11R)-5Ja agreed well with the experimental spectrum and confirmed the (4R, 5R, 6S, 7R, 8R, 9R, 10R, 11R) absolute configuration. Based on biosynthetic considerations [4,13], similar ECD data of 6 and 5 revealed the same absolute configuration of 6 as that of 5. Thus, the structures of compounds 5-6 were established, as shown in Figure 2, named 11-methoxymethylcardivarolide K and 11-methoxymethylcardivarolide L, respectively. The HRESIMS data of compounds 7-8 suggested the molecular formulas of C26H40O10 and C26H38O10, respectively. The NMR data of 7 were similar to those of 5, except that an angeloyloxy group appeared in 7 instead of a 3-methylbutyryloxy group at C-5 in 5. For the same reason, the NMR data implied the presence of an angeloyloxy group at C-9 rather than a 3-methylbutyryloxy group in 8 compared to 7. The 1 H− 1 H COSY, HSQC, and HMBC spectra of 7-8 confirmed these observations, leading to the assignment of their The HRESIMS data of compounds 7-8 suggested the molecular formulas of C 26 H 40 O 10 and C 26 H 38 O 10 , respectively. The NMR data of 7 were similar to those of 5, except that an angeloyloxy group appeared in 7 instead of a 3-methylbutyryloxy group at C-5 in 5. For the same reason, the NMR data implied the presence of an angeloyloxy group at C-9 rather than a 3-methylbutyryloxy group in 8 compared to 7. The 1 H− 1 H COSY, HSQC, and HMBC spectra of 7-8 confirmed these observations, leading to the assignment of their planar structures. The relative configurations of 7-8 were deduced to be the same as 1 on the basis of similar ROESY data. Considering similar ECD data of 7-8 and 1 resulted in the conclusion of their same absolute configurations. Due to the fact that there are some differences in the ECD spectra of 1 and 7, the absolute configuration of 7 was further confirmed by ECD calculations. Similarly, a simplified structure named 7Ja (Supporting Information C1), in which an acetyl group instead of a 3-methylbutyryloxy moiety was used for ECD calculations [16]. It was clear that the calculated ECD spectrum of (4R, 5R, 6S, 7R, 8R, 9R, 10R, 11R)-7Ja was matched very well with the experimental ECD spectrum of 7 ( Figure 6). Thus, the structures of 7-8 were elucidated and were named 11-methoxymethylcardivarolide G and 11-methoxymethylcardivarolide F.
Molecules 2022, 27, x FOR PEER REVIEW 7 of 13 planar structures. The relative configurations of 7-8 were deduced to be the same as 1 on the basis of similar ROESY data. Considering similar ECD data of 7-8 and 1 resulted in the conclusion of their same absolute configurations. Due to the fact that there are some differences in the ECD spectra of 1 and 7, the absolute configuration of 7 was further confirmed by ECD calculations. Similarly, a simplified structure named 7Ja (Supporting Information C1), in which an acetyl group instead of a 3-methylbutyryloxy moiety was used for ECD calculations [16]. It was clear that the calculated ECD spectrum of (4R, 5R, 6S, 7R, 8R, 9R, 10R, 11R)-7Ja was matched very well with the experimental ECD spectrum of 7 ( Figure 6). Thus, the structures of 7-8 were elucidated and were named 11-methoxymethylcardivarolide G and 11-methoxymethylcardivarolide F. Figure 6. Experimental ECD spectrum of 7 and calculated ECD spectra of 7Ja and 7Jb.

Analysis of the Macrophages Culture Supernatants PGE2 Levels
Numerous studies have suggested the biologically pivotal roles of PGE2 in cancer, inflammation, and pain [17][18][19][20][21]. Due to the insufficient amount of isolates, only compounds 2, 3, and 8 were tested for the effects on PGE2 production in the supernatant of LPS-induced RAW 264.7 cells by a highly sensitive ELISA in this study. LPS stimulation resulted in a marked increase in PGE2 in the macrophage culture supernatants or the mice sera. Among the three compounds, pretreatment with 8 could potently decrease PGE2 contents, even lower than the normal level ( Figure 7).

Analysis of the Macrophages Culture Supernatants PGE2 Levels
Numerous studies have suggested the biologically pivotal roles of PGE2 in cancer, inflammation, and pain [17][18][19][20][21]. Due to the insufficient amount of isolates, only compounds 2, 3, and 8 were tested for the effects on PGE2 production in the supernatant of LPS-induced RAW 264.7 cells by a highly sensitive ELISA in this study. LPS stimulation resulted in a marked increase in PGE2 in the macrophage culture supernatants or the mice sera. Among the three compounds, pretreatment with 8 could potently decrease PGE2 contents, even lower than the normal level ( Figure 7). Figure 7. Analysis of the macrophages culture supernatants prostaglandin E2 levels by a highly sensitive ELISA, when treated with compounds 2, 3, and 8. ** p < 0.01 vs. control group, ## p < 0.01 vs. model group.

Plant Material
The whole plant of C. divaricatum was collected from EnShi, Hubei province of China

X-ray Crystal Structure Analysis
X-ray diffraction data were collected on the Agilent GEMINITME instrument (CrysAl-isPro software, Version 1.171.35.11), with enhanced Cu Kα radiation (λ = 1.54184 Å). The structure was solved by direct methods and refined by full-matrix least-squares techniques (SHELXL-97). All non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were located by geometrical calculations and from positions in the electron density maps. Crystallographic data (excluding structure factors) for 1 in this paper have been deposited with the Cambridge Crystallographic Data Centre (Deposition Number: CCDC 1846500). Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-12-23336033 or e-mail: deposit@ccdc.cam.ac.uk).
Cell viability assay: The assay was run in triplicate. In a 96-well plate, each well was plated with 2 × 10 4 cells. After cell attachment overnight, the medium was removed, and each well was treated with 100 µL of medium containing 0.1% DMSO or different concentrations of the test compounds and the positive control cis-platin. The plate was incubated for 4 days at 37 • C in a humidified, 5% CO 2 atmosphere. Cytotoxicity was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [22]. After addition of 10 µL MTT solution (5 mg/mL), cells were incubated at 37 • C for 4 h. After adding 150 µL DMSO, cells were shaken to mix thoroughly. The absorbance of each well was measured at 540 nm in a multiscan photometer. The IC 50 values were calculated by Origin software.
The culture supernatant assay: For the culture supernatant assay, RAW 264.7 cells were pretreated with the tested compounds (10 µM) for 2 h and then stimulated with LPS (10 µg/L) for 24 h. PGE2 concentrations in the culture supernatants were simultaneously assayed by hscELISA. At least 10-and 50-fold dilutions are needed for the culture supernatant tests.
Statistical analysis: Values were expressed as mean ± SD. Statistical analyses were performed using the Student's t-test. Differences were considered significant when associated with a probability of 5 % or less (p ≤ 0.05).

Conclusions
In conclusion, eight new compounds (1)(2)(3)(4)(5)(6)(7)(8) representing a new subtype (subtype V, named 3-oxo-11-methoxymethylgermacranolide) of germacranolides, were isolated from the whole plant of C. divaricatum. Notably, a pair of isomers (5/6) was obtained from the same plant. The absolute configuration of compound 1 was unambiguously established by X-ray diffraction. The other compounds with the same skeleton were determined by comparison of NOESY and ECD data with those of 1. Structurally, all compounds contained a 5-membered γ-lactone ring with the methoxymethyl group fused to a circular 10-membered carbocycle. Based on the common structural features, these germacranolide analogs are different as far as substituents are concerned. Compound 4 showed weak cytotoxicity against a human tumor cell line. Compound 8 could potently decrease PGE2 productions in LPS-induced RAW 264.7 cells. These findings are an important addition to the present knowledge on the structurally diverse and biologically important germacranolide family.
Author Contributions: T.Z. performed the isolation and identification of all the compounds and also wrote this paper, H.Z., C.L. and L.F. contributed to biological activity evaluation and wrote this section. Z.Z. designed and guided the experiment and also revised the paper. All authors have read and agreed to the published version of the manuscript.