Three new cardenolides from the fruits of Cascabela thevetia (L.) Lippold and their cytotoxic activities

Abstract Phytochemical investigations on the fruits of Cascabela thevetia (L.) Lippold led to obtain three new cardenolides (1–3) and five known analogues (4–7). Their structures were elucidated by means of UV, IR, HR-ESI-MS, 1D and 2D NMR spectroscopic data analysis. Compounds 1 and 2 represent the first examples of naturally occurring cardenolides with 19-nor-5(10)-ene group and α-l-3-demethyl-thevetose, respectively. Compound 3 is a rare C-nor-D-homocardenolide in nature. All isolated cardenolides (1–7) were evaluated for their cytotoxic activities against four human cancer cell lines (MCF-7, HCT-116, HeLa and HepG2), and the results indicated the compounds with sugar units (1, 2, 4, and 5) exhibited stronger cytotoxic activities with IC50 values ranging between 0.022 and 0.308 μM. GRAPHICAL ABSTRACT


Introduction
Cardenolides have been traditionally used for the treatment of congestive heart failure and arrhythmias (Schoner and Scheiner-Bobis 2007;Tian et al. 2008;Kolkhof et al. 2010).Recently, Epidemiological data, along with the results obtained from in vitro and in vivo studies showed that they exhibit potent antiproliferative and apoptotic effects on cancer cells through complex signal transduction mechanisms (Nasu et al. 2005;Rimpelov a et al. 2021).Therefore, cardenolides may represent a promising chemical entity for in cancer chemotherapy.Cascabela thevetia (L.) Lippold (synonym: Thevetia peruviana (Pers.)K. Schum) belongs to Apocynaceae family and widely grows in the tropics and subtropics, which has been in folk medicine with antipyretic, rodenticide, and therapeutics for heart failures (Alonso-Castro et al. 2011).Previous chemical investigations have demonstrated that the whole plant of C. thevetia contained rich cardenolides, particularly its seeds (Cheng et al. 2016;Tian et al. 2016).However, as part of our continuous effort to search for novel cardenolides with antitumor effects (Li et al. 2014;2018), seven cardenolides (1-7), including three new compounds (1-3) (Figure 1), were obtained from the fruits of C. thevetia.Herein, we describe the isolation and structural elucidation of these new compounds.Additionally, the cytotoxic activities of these isolated compounds (1-7) toward human cancer MCF-7, HCT-116, HeLa and HepG2 cell lines were evaluated by MTT method.group (218 nm) and the IR spectrum displayed an absorption band for c-lactone carbonyl (1734 cm À1 ), revealing the presence of a butyrolactone system in 1 (Tian et al. 2016).The 1 H and 13 C NMR signals for 1 were assigned based on 1 D and 2 D NMR experimental results (Table S1).), 18.9 (C-6 0 ), and 61.0 (-OCH 3 ), respectively.In additional, the 13 C NMR resonances at d C 99.7 (C-1 0 ), 74.1 (C-2 0 ), 85.8 (C-3 0 ), 76.9 (C-4 0 ), 69.3 (C-5 0 ), 18.9 (C-6 0 ), and 61.0 (-OCH 3 ) revealed the presence of L-thevetose moiety in 1, which was also supported by comparison of its observed spectroscopic data with those of reported (Abe et al. 1994;Miyagawa et al. 2009;Tian et al. 2016).The a-orientation of the L-thevetose moiety was defined by the small coupling of H-1 0 (J ¼ 3.9 Hz) (Inoue et al. 2000;Pei et al. 2011;Li et al. 2018;2022).Moreover, the HMBC correlation of H-1 0 with C-3 (d C 73.9) confirmed that the L-thevetose moiety was located at C-3 position of the aglycone.Consequently, the structure of compound 1 was determined as 19-nor-5(10)-en-digitoxigenin 3b-O-a-L-thevetoside, and named castheveside A.

Results and discussion
Compound 2 was obtained as a white amorphous powder.Its molecular formula was assigned as C 29 H 44 O 8 by the 13 C NMR and HR-ESI-MS (m/z 521.3116 .3114) data, with 8 degrees of unsaturation.The IR spectrum suggested the presence of hydroxyl groups (3444 cm À1 ) and carbonyl of an c-lactone carbonyl moiety (1758 cm À1 ).The 1 H and 13 C NMR signals for 2 were assigned based on 1 D and 2 D NMR experimental results (Table S1), which showed a typical signals of cardenolide skeleton.The 1 H NMR spectrum of 2 showed characteristic signals of a unsaturated lactone unit at d H 5.04 (1H, dd, J ¼ 18.0, 1.2 Hz, H-21a), 5.33 (1H, dd, J ¼ 18.0, 1.2 Hz, H-21b), and 6.14 (1H, br s, H-22), along with two methyl groups at d H 1.02 (3H, s, H-18) and 0.81 (3H, s, H-19), indicating the aglycone of 2 to be digitoxigenin (Drakenberg et al. 1990).Furthermore, the 1 H and 13 C NMR spectra showed an anomeric proton doublet at d H 5.33 (1H, d, J ¼ 3.9 Hz, H-1 0 ), one methyl proton doublet at d H 1.66 (3H, d, J ¼ 6.3 Hz, H-6 0 ) as well as carbon signals at d C 99.4 (C-1 0 ), 74.6 (C-2 0 ), 75.7 (C-3 0 ), 78.1 (C-4 0 ), 69.5 (C-5 0 ), and 19.1 (C-6 0 ), revealing that the presence of a 6deoxyhexose in 2. Comparison of the 1 H and 13 C NMR data of the 6-deoxyhexose moiety in 2 with those of L-thevetose in 1 or other related compounds (Abe et al. 1994;Miyagawa et al. 2009;Tian et al. 2016) indicated that they had the similar structure except for the absence of one methoxyl carbon signal at d C 61.0 (1).The upfield shift of C-3 0 from d C 85.8 in 1 to d C 75.7 in 2, and the downfield shift of C-2 0 and C-4 0 from d C 74.1 and 76.9 in 1 to d C 74.6 and 78.1 in 2, suggesting that the 6-deoxyhexose moiety of 2 was a 3-demethylated derivative of L-thevetose.The a-orientation of the sugar moiety was defined by the coupling constants of H-1 0 (J ¼ 3.9 Hz).Moreover, the HMBC (Figure S1) correlation of H-1 0 with C-3 (d C 74.1) confirmed that the sugar moiety was located at C-3 position of the aglycone.Thus, compound 2 was determined as digitoxigenin 3b-O-a-L-3-demethyl-thevetoside, and named castheveside B.
Compound 3 was isolated as a white amorphous powder.The molecular formula of 3 was determined as C 23 H 32 O 4 by the HR-ESI-MS at m/z 373.2379  -18), indicating that 3 had a C-nor-Dhomocardenolide skeleton (Abe et al. 1992a(Abe et al. , 1992b)).Analyses of the 1 H and 13 C NMR data with those of thevetioside A (Abe et al. 1992a) indicated that 3 had the similar structure as the aglycone of thevetioside A except for obvious differences of ring A (i.e. the downfield shift of C-1, C-2, C-4, and C-5 from d C 32.5, 26.9, 30.8, and 36.5 in thevetioside A to d C 37.6, 31.5, 37.8, and 42.9 in 3), suggesting that 3 was a C-3 positon stereoisomer of thevetiogenin (Abe et al. 1994).Furthermore, the carbon signals of ring A in 3 (C-1$C-5 and C-10) had highly consistent with those of 3a, 5b-dihydroxyl cardenolides (Zhu et al. 2018;Bedir et al. 2021), and the ROESY (Figure S1) correlation of H-3 (d 3.51, 1H, m) with H-5 (d 1.48, 1H, m) indicated that H-3 of 3 was a-oriented.Thus, the structure of 3 was assigned as 3a-thevetiogenin.
The cytotoxicity of compounds 1-7 against human cancer MCF-7, HCT-116, HeLa and HepG2 cell lines were evaluated by MTT assay.Colchicine was used as a positive control.The results showed that compounds 1, 2, 4, and 5 (with sugar unit) exhibited significant inhibitory activity against the four cell lines with IC 50 values ranging between 0.022 and 0.308 lM, whereas compounds 3, 6, and 7 (without sugar unit) exhibited no inhibitory activity with initial screening concentration at 10 lM.Therefore, these results further suggest that the presence of sugar unit at the C-3 of cardenolides is not only an important group to improve the physiological properties of molecules, but also a main pharmacophore to enhance their anticancer effect (Xue et al. 2013;2014;Li et al. 2018;Du et al. 2021).

General experimental procedures
Optical rotations were obtained on a P-1020 digital polarimeter.UV spectra were measured with a JASCO V-550 UV/vis spectrophotometer.IR spectra were recorded on a JASCO FTIR-480 plus spectrometer.1D and 2D NMR spectra were recorded on a Bruker AV 300, 400 and 600 spectrometers at room temperature.The chemical shift (d) values are expressed in ppm relative to the chemical shifts of solvent resonances (pyridine-d 5 : d H 7.58 and d C 135.8 ppm; CD 3 OD: d H 3.31 and d C 49.0 ppm) with TMS as internal standard, and coupling constants (J) were in hertz (Hz).HR-ESI-MS and ESI-MS data were obtained on a Micromass Q-TOF and SCIEX Triple Quad 4500 mass spectrometer, respectively.Analytical HPLC was performed on Agilent Technologies 1260 infinity II liquid chromatography system equipped with an SPD-M20A diode array detector using an Agilent C 18 column (4.6 Â 100 mm, 5 lm) or an Epic C 18 column (4.6 Â 100 mm, 5 lm).Semipreparative HPLC was acquired on Agilent Technologies 1260 infinity liquid chromatography system equipped with quaternary pump, autosampler, DAD detector using an Epic C 18 semipreparative column (10 Â 250 mm, 5 lm).Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao Haiyang Chemical Group Corp, Qingdao).TLC analysis was performed on pre-coated silica gel GF254 plates (Qingdao Haiyang Chemical Group Corp, Qingdao).

Plant material
The fresh fruits of C. thevetia were collected from Xuwen county, Zhanjiang City of Guangdong province, P.R.China (altitude: 100 $ 150 meters; longitude: 109 55 0 , latitude: 20 15 0 ) in July 2020, and authenticated by Prof. Z. P. Gou (Guangdong Medical University) .The voucher specimen (No.T202007) had been deposited at the School of Pharmacy, Guangdong Medical University.