New Terpenoids And Lignans From The Twigs Of Tripterygium Hypoglaucum

Abstract Six new compounds, including three terpenoids (1-3) and three lignans (4-6), were isolated from the 95% EtOH extract of the twigs of Tripterygium hypoglaucum. Their structures were determined on the basis of extensive spectroscopic analysis. 9′-O-benzoyl-lariciresinol (4) showed weak cytotoxicity against HepG2/Adr cells, with an IC50 value of 30.1 μM in vitro. GRAPHICAL ABSTRACT

T. hypoglaucum is mainly distributed in China and East Asia (Li et al. 2015). It has been used for the treatment of rheumatoid arthritis, muscle and skeletal injury, as well as skin disorders in Chinese folk medicine (Tao and Lipsky 2000). Previous chemical studies have showed that T. hypoglaucum characteristically contained sesquiterpenes Zhao et al. 2014), diterpenoids (Fujita et al. 2000), triterpenes (Zhao et al. 2018). In the course of our search for the anti-tumor agents from Tripterygium plants, 2a-hydroxy-triptolide from T. hypoglaucum was found to show significant cytotoxic activity against A549, KB, KBvin, and MDA-MB-231 cell line (Li et al. 2015). A further chemical investigation on this plant led to the isolation of six new compounds. In this paper, we described the isolation, structure elucidation, and cytotoxicity of the six new compounds.
Compound 1 was isolated as colorless oil. Its molecular formula was determined as C 19 H 28 O 6 from its positive HRESIMS spectrum (m/z 375.1784 [M þ Na] (1 H,d,J ¼ 15.8 Hz,. The 13 C NMR and DEPT spectrum of 1 showed signals for 19 carbons, including five methyls, five methylenes, three methines, and six quaternary carbons (Table S1 in the Supplementary Material). Detailed comparison of 1 H and 13 C NMR data of 1 with 3b-hydroxy-5a,6a-epoxy-7-megastigmen-9-one (Duan et al. 2002) indicated that the two compounds were structurally similar, except for the only difference of the substituent at C-3.
The molecular formula of 2 was established to C 17 H 24 O 6 from its positive HRESIMS spectrum (m/ (1 H, s)]. The 13 C NMR spectrum showed the signals for four methyls [d C 14.5 (C-6 0 ), 26.5 (C-9), 27.1 (C-11), 30.8 (C-10)], five methylenes [d C 29.8 (C-3 0 ), 30.3 (C-2 0 ), 43.6 (C-4), 45.1 (C-2), 61.8 (C-5 0 )], one methane [d C 114.1 (C-7)], and six quaternary carbons including three carbonyls [d C 173.1 (C-1 0 ), 174.1 (C-8/C-4 0 )], an olefinic (d C 174.1, C-6) and two aliphatic quaternary carbons [d C 36.9 (C-1), 87.9 (C-5)] (Table S1 in the Supplementary Material). A comparison of 1 D NMR spectra between 1 and 2 suggested that 2 also contained the monoethyl succin- The 1 H and 13 C NMR spectroscopic data of 2 was similar to those of loliolide (Tanaka and Matsunaga 1989). The significant downfield shift of H-3 (d H 5.26) and C-3 (d C 70.5) in 2 relative to that of loliolide (d H 4. 33 and d C 66.8), implied that the OH at C-3 was esterified by the monoethyl succinate. The relative configuration of 2 was established by the ROESY spectrum. The ROESY correlations of Me-9/Me-11 and Me-10/H-3 indicated the b-orientation of Me-11 and the a-orientation of H-3. Consequently, the structure of 2 was assigned and named as loliolide A.  (1 H, m)]. The 13 C NMR and DEPT spectrum of 3 showed signals for 21 carbons, including a tetrasubstituted-benzene C-atom signals [d C 155.4 (C-12) Table S1 in the Supplementary Material). The spectral characteristics and consideration of the isolated from the plants suggested that 3 was an abietane-typediterpene (Li et al. 2015). The spectral data of 3 was very similar to those of triptobenzene R (Li et al. 2013), except for the presence of one more methoxy in 3. This methoxy was assigned to C-12 as indicated by the HMBC correlation from the methoxy to C-12. The relative configuration was determined by the analysis of ROESY spectrum. ROESY correlations between H a -19/Me-20 and Me-18/H-5 indicated the b-orientation of H 2 -19. Thus, the structure 3 was established and named triptobenzene W.
The molecular formula of 4 was determined as C 27 H 28 O 7 from its positive HRESIMS spectrum (m/z 487.1727 (Table S2 in the Supplementary Material). All the spectral data indicated that 4 was a tetrahydrofurantype lignan. The 1 H and 13 C NMR (Table S2 in the Supplementary Material) data of 4 were very similar to those of agastinol (Lee et al. 2002), except for the absence of hydroxyl group at position C-4 00 in 4. The above observation suggested that the phydroxybenzoic acid was displaced by a benzoic acid, which was confirmed by the HMBC correlations of H-2 00 with C-1 00 , C-4 00 , C-6 00 , and C-7 00 , H-4 00 with C-2 00 and C-6 00 , and H-9 0 with C-7 00 . ROESY correlations of H-9 0 /H-7, H-8/H-8 0 , and H-7 0 /H-9 0 indicated b-orientation for H-7 0 and a-orientation for H-8 and H-8 0 ( Figure S2 in the Supplementary Material). Consequently, the structure of 4 was determined to be 9 0 -Obenzoyl-lariciresinol.
Compound 5 was obtained as white amorphous powder. Its molecular formula was established to be C 28 H 30 O 8 concluded from its positive HRESIMS spectrum (m/z 517.1845 [M þ Na] þ , calcd 517.1833) with fourteen degrees of unsaturation. Analyses of the 1 H and 13 C NMR data suggested that the structure of 5 was very similar to those of 4 (Table S2 in the Supplementary Material). The only difference was the presence of one more methoxy group in 5. The methoxy group was deduced to attach to C-5 0 which was confirmed by the HMBC correlation from OMe to C-5 0 . The relative configuration of 5 was deduced to be identical to those of 4 due to similarity of ROESY spectrum. Therefore, 5 was elucidated as 9 0 -O-benzoyl-5 0 -methoxylariciresinol.
Compound 6 was isolated as white amorphous powder. Its molecular formula was determined as C 17 H 24 O 6 inferred from its positive HRESIMS spectrum (m/z 529.1615 [M þ Na] þ , calcd 529.1623) with fifteen degrees of unsaturation. The 1 H and 13 C NMR data ( Table S2 in (1 H, d, J ¼ 16.0) at C-9 0 in 6 by the benzoyl in 4, which was supported by HMBC correlation from H-9 0 (d H 4.32) to the carbonyl carbon (d C 166.9). The relative configuration of 6 was established to be identical to those of 5 due to the similarity of ROESY spectrum between them. Thus, the structure of 6 was identified as 9 0 -O-cinnamoyl-lariciresinol.

Biological studies of compounds 1-6
Compounds 1-6 were tested for in vitro cytotoxic activity against HepG2/Adr cells. In consequence, all tested compounds were inactive except that 9 0 -O-benzoyl-lariciresinol (4) showed weak cytotoxicity with an IC 50 value of 30.1 lM.

Plant material
The twigs of T. hypoglaucum were collected in Cang Mountain, Dali, Yunnan Province, People's Republic of China, in July 2015. The sample was identified by Prof. Bei Jiang, University of Dali. A voucher specimen (KMUST201507) has been deposited at the Laboratory of Phytochemistry, the Faculty of Life Science and Technology, Kunming University of Science and Technology.

Extraction and isolation
The air-dried twigs (35 kg) of T. hypoglaucum were extracted with 95% EtOH (3 Â 60 L, 24 h, each). The extracts were combined and concentrated under reduced pressure to a crude extract (5 kg). The crude extract was suspended in water and then partitioned sequentially with petroleum ether, EtOAc, and n-BuOH, respectively.