Structure modification and anti-tumor activity evaluation on sesquiterpene lactones TBA and TBB from Sphagneticola trilobata

Abstract Thirteen sesquiterpene lactone derivatives were designed and synthesised using the sesquiterpene lactone epimers TBA and TBB as the parent molecules, isolated from Sphagneticola trilobata. Their structures were elucidated by extensive spectroscopic studies and chemical evidence. Furthermore, the structures of 1a, 4b and 5a were confirmed by X-ray single-crystal diffraction analyses. All of the compounds were further examined for their in vitro antiproliferative activity against human cancer cell lines HeLa and MCF-7. Unfortunately, the activity of all derivatives were weaker than that of the parental compounds. Graphical Abstract


Introduction
Cancer is a complex disease which has seriously threatened human life, health and social development.According to International Agency of Cancer Research (IARC) the burden cancer incidence and mortality is growing worldwide.Based on GLOBOCAN 2020, 18.1 million new cancer cases (except nonmelanoma skin cancer) occurred in 2020 (Sung et al. 2021).Natural sesquiterpene lactones (SLs) are a family of secondary metabolites derived almost exclusively from Asteraceae/Compositae plants (Li et al. 2016).These natural products have been a hot topic in the field of tumor drugs research because of the complexity, diversity, novelty of structures and the uniqueness of function (Ren et al. 2016).For instance, the a-santonin-derived analogues hold promise as chemotherapeutic agents for the treatment of human hepatocellular cancer (Chen et al. 2018).The dithiocarbamate esters, SAHA and 5-fluorouracil were added to parthenolide, and extensive research work has been carried out to characterise the anticancer activity, the structure-bioactivity relationship (SAR), and the molecular mechanisms (Ding et al. 2018;Ding et al. 2019;Ge et al. 2019).Alantolactone had demonstrated potential anticancer activity, and the new amino scaffolds of the alantolactone could improve their water solubility (Kumar et al. 2018).SLs' cytotoxic activity has been ascribed as mainly dependent on the a-methylene-c-lactone group, which is prone to react with suitable nucleophiles (Kupchan et al. 1971;PoloL et al. 2007;R o_ zalski et al. 2007).
Sphagneticola trilobata (Wedelia trilobata) is a perennial herb belonging to the genus Sphagneticola of the Asteraceae family.It is listed as one of the 100 worst invasive species around the world.It was first introduced in Hong Kong in the 1870s to replace the traditional Chinese medicine S. calendulacea for the treatment of diphtheria, whooping cough, dysentery, hemorrhoids and bruises (Sun et al. 2020).Trilobolide-6-O-isobutyrate A and B (TBA, and TBB), a pair epimer of SLs with high oxygen content were isolated from the flowers of S. trilobata, they also have a a-methylene-c-lactone group.Furthermore, the absolute configuration of TBA and TBB were determined by X-ray single-crystal diffraction analyses (Hui et al. 2018).Subsequent investigations indicated that TBB could strongly inhibit the proliferation of hepatocellular carcinoma through inhibition of the IL-6/STAT3 signaling pathway (Zhou et al. 2021).Inspired by the structure modification of parthenolide and other SLs, the structure modification of TBA and TBB were conducted further and the antitumor activity of all the derivatives were evaluated.

Chemistry
Trilobolide-6-O-isobutyrate A and B (TBA, and TBB) were isolated from the EtOH solutions of the dried flowers of S. trilobata by repeated column chromatography and these two compounds were further purified by recrystallisation from EtOH as previously described methods (Hui et al. 2018).Schemes 1 and 2 represents the synthetic pathways of all the compounds, respectively.First, the appropriate sesquiterpene lactones and CaH 2 solid were dissolved in the MeOH to the chromatography using CHCl 3 -EtOAc gradient to afford pure product (compounds 1a, 1b).KOH was used to provide alkaline conditions and acetone as solvent, the reaction mixture was stirred at room temperature for 8.5 h.The extracted organic solution was subjected to column chromatography to afford pure product (compounds 2a, 2b-3b) (Li et al. 2013;Schirmeister et al. 2017;Rong et al. 2020).In order to obtain different degrees of removal of the ester groups at C-1, C-6, and C-9 positions and expose the hydroxyl groups, we switched to using KOH to provide alkaline conditions and MeOH as the solvent.The reaction mixture was stirred at room temperature for 3.5 h.Then, crude products were further purified by silica gel column chromatography to afford pure product (compounds 3a-5a, 4b-6b).According to the above method, the hydrolysis time of TBA or TBB was extended to 4.5 h, and the pure product (6a, 7b) was prepared by HPLC.
The structures of these derivatives were firmly established by combination with the physical and chemical properties and 1D/2D NMR, HRMS methods.The Michael addition at the a-methylene-c-lactone of SLs with amines have been found to be highly stereo specific and yielded exclusively a single stereo isomer with the (R Ã )-configuration at the newly formed C-11 chiral carbon.This was attributed to the stereoexclusive protonation of the enolate formed during Michael addition (Wang et al. 2015).In this research, an NOE effect between H-11 and H-6 confirmed the 11 R stereo chemistry of SLs derivatives, while the X-ray crystal structure of 1a, 5a and 4b (Figure 1) further confirmed the R configuration at C-11.

In vitro anticancer activity
The anticancer activity of TBA, TBB, and their derivatives was evaluated by the MTT assay against human tumor cell lines HeLa and MCF-7 and compared with Adriamycin as the working standard.The final inhibitory concentration, suppressing cell growth by 50%, was calculated using the reported methods (Ren et al. 2020).As shown in Table 1, all derivatives showed comparatively high IC 50 values compared to TBA and TBB.Compound 4a demonstrated the best antiproliferative activity against HeLa cells with an IC 50 value of 27.83 mM.On the basis of the above activity data, a preliminary SAR was deduced.Compounds 4a and 5b with hydroxy at C-6 exhibited stronger effects on HeLa and MCF-7 cells than corresponding compounds 1a and 1b bearing isobutyryl.In addition, compared with compounds 5a and 6a, 6b and 7b, the hydroxy at C-4 was essential for the activity.Notably, introduction of a methoxy or prop-2carbonyl group at C-13 position afforded all compounds, which were less potent than that of the parental compounds, and further confirmed SLs' cytotoxic activity has been ascribed as mainly dependent on the a-methylene-c-lactone group (Kupchan et al. 1971;PoloL et al. 2007;R o_ zalski et al. 2007).

Table 1 .
Antiproliferative activity of all the compounds against two human cancer cell lines.