Noncytotoxic 16,23-epoxycucurbitacin-type triterpenoids from Elaeocarpus hainanensis

Abstract Phytochemical investigation of methanol extract from Elaeocarpus hainanensis Oliv. leaves and twigs led to the isolation and structural determination of three 16,23-epoxycucurbitacin-type triterpenoids, including a new hydroperoxide, 16α,23α-epoxy-3β,20β-dihydroxy-24α-hydroperoxy-10αH,23βH-cucurbit-5,25-dien-11-one (elahainencin A, 1), and two known analogs (2 and 3). Their chemical structures were determined by the spectroscopic analyses, including 1 D-, 2 D NMR and HR ESI MS spectra. Compound 1 represents a cucurbitacin derivative incorporating a hydroperoxide. In addition, these isolated compounds have been found to be noncytotoxic when tested in vitro against five human cancer cell lines (A549, T24, 8505, Huh-7 and SNU-1) by using the SRB method. Graphical Abstract

Elaeocarpus hainanensis Oliv.widely distributed in China, Laos, Myanmar, Thailand, and Vietnam.Previous studies revealed that this species contains a variety of triterpenoids, including novel cucurbitacins (Meng et al. 2008), oleanane-type triterpenes, and their glycosides (Nga et al. 2018;Tung et al. 2020).As part of our ongoing study on E. hainanensis growing in Vietnam, we report herein the isolation and structural characterization of elahainencin A (1), a new 24a-hydroperoxy-16,23-epoxycucurbitacin as well as two other known analogues 2 and 3.
Compound 1 was obtained as a white solid.Its molecular formula was assigned as C 30 H 46 O 6 on the basis of the quasi-molecular ion peaks at m/z 503.3358S1).Thus, the planar structure of 1 was established on the basis of 13 C-1 H correlation signals in HSQC and HMBC spectra.
The absolute configuration at C-24, hydroperoxide functional group, could not be established based on the NOESY data due to free rotation.However, the value of J coupling constant between H-23 and H-24 (J H-23/H-24 ¼ 7.5 Hz) suggested the relative threo-configuation of disubstituted groups at C-23 and C-24.Except for the signals of side chain from C(24) to C(27), the 1 D-and 2 D NMR data of 1, including identical corresponding signals and typical coupling constant values, indicated the corresponding identical resemblance to those of 16a,23a-epoxy-3b,20b-dihydroxy-10aH,23bH-cucurbit-5,24-dien-11-one (2) and its 3-O-b-D-glucopyranoside (3), the known epoxycucurbitacins isolated from the same extract in present study, also found in this species, E. chinensis and E. petiolatus (Meng et al. 2008;Pan et al. 2012;Cho et al. 2022).Therefore, the relative configuration at all asymmetric carbons of 1 were determined as shown in the Figure 1.In the above-mentioned data, the structure of 1 was elucidated as 16a,23a-epoxy-3b,20b-dihydroxy-24a-hydroperoxy-10aH,23bH-cucurbit-5,25dien-11-one, a new natural cucurbitacin, and trivially named elahainencin A. All 1 Hand 13 CNMR data of 1 were shown in Table S1 on the basis of HSQC, HMBC spectra and comparison with 2 and 3 (Table S1, Supporting Information).
The co-occurrence of 1, 2 and 3 in the same plant material suggests that 1 might be originated through isomerization of the double bond C 24 -C 25 , accompanied by peroxidation during biosynthesis (Seger et al. 2004).To the best of our knowledge, elahainencin A is the first example of cucurbitacin hydroperoxide.
Isolated compounds 1-3 were evaluated for their cytotoxicity on carcinoma cell lines, including A549, T24, 8505, Huh-7 and SNU-1 cells.All three tested compounds (1-3) exhibited no obvious cytotoxicity up to IC 50 > 50 mM (Table S2, see details in 471.3469), originating from a molecular ion after loss of two oxygen atoms (O 2 ) and characteristic for the presence of a hydroperoxide group in the parent compound (Shang et al. 2020).Moreover, 13 C NMR data of 1 were almost similar to those of elaeocarpucins A and B (Pan et al. 2012), with the only significant downfield shift difference at d C 93.2 (Dd C > 25 ppm), which confirmed the assignment of hydroperoxide group at C-24.Furthermore, key HMBC correlations from H-24 (d H 4.22) and H-27 (d H 1.75) to C-26 (d C 116.2), as well as the terminal olefinic methylene protons of H 2 -26 to C-24 (d C 93.2), C-25 (d C 143.7), and C-27 (d C 18.0) confirmed the structure of the side chain moiety (Table
The 1 H-and 13 C-NMR spectra revealed the presence of seven singlet methyl groups, a doublet olefinic methine at d H 5.66 (br d, J ¼ 6.0 Hz, H-6), and four oxygenated methines at d H 3.45 (brs, H-3)/d C 76.9, d H 4.32 (ddd, J ¼ 10.5, 10.5, 3.5 Hz, H-16)/d C 78.0, d H 3.92 (m, H-23)/d C 76.2, and d H 4.22 (d, J ¼ 7.5 Hz, H-24)/d C 93.2.The HMBC analysis revealed the presence of three prominent fragments, -C 2 -C 3 -C 4 -, -C 15 -C 16 -C 17and -C 23 -C 24 -C 25 -C 27 -.The characteristic NMR spectral data of 1 were similar to those of 503.3367) and 537.2989 [M þ Cl] -(calcd.for C 30 H 46 ClO 6 -, 537.2988) in the positive and negative HR ESI MS, respectively.The 13 C NMR spectrum of 1 revealed the presence of 30 carbons, which were classified from HSQC data as eight quaternary carbons, eight methines, seven methylene and seven methyl groups (8 x C, 8 x CH, 7 x CH 2 , 7x CH 3 ), including five oxygen-bearing carbons, a trisubstituted double bond, a disubstituted terminal double bond, and a carbonyl group (d C 216.6).C 143.7 (C-25).Moreover, the olefinic methine signal at C-24 in 2 were replaced with an oxymethine signal (d H 4.22/d C 93.2) in 1.The substantial downfield shift observed for these resonances suggested the presence of a hydroperoxide group.This was confirmed by the base peak in the positive ion HR ESI MS spectrum at m/z 471.3479 [