Four New Picrotoxane-Type Sesquiterpenes From Dendrobium nobile Lindl

Four picrotoxane-type sesquiterpenes, dendroterpene A–D (1–4), together with four known compounds (5–8), were isolated from the stems of Dendrobium nobile Lindl. Their structures were elucidated by spectroscopic analysis, X-ray diffraction analysis, analysis of the ECD data according to the Klyne's lactone sector rule, and quantum ECD calculation. Compounds 1 and 2 are two new picrotoxane-type sesquiterpenes with a new carbon skeleton containing a formamide group, which may be derived from the previously reported dendrobiumane B skeleton by the C(9)-C(11) carbon bond cleavage. Compounds 3, 5, 6, and 8 exhibited inhibitory activity against α-glycosidase. Compounds 5 and 6 were cytotoxic against SGC-7901, K562, A549, BEL-7402, and Hela cell lines.


INTRODUCTION
Dendrobium nobile Lindl, the plant belongs Orchidaceae family, is one of three major plant sources of the traditional Chinese medicine "Shi Hu" in Chinese (edition 2005), which are used as a tonic to nourish the stomach and promote the production of body fluid recorded in the Chinese Pharmacopeia (Jiangsu New Medical College, 1986). The previous reports showed a series of chemical constituents isolated from D. nobile including sesquiterpenes (Zhang et al., 2007a), alkaloids (Liu and Zhao, 2003;Meng et al., 2017), bibenzyls derivatives (Zhang et al., 2007b), and glucosides (Zhao et al., 2001), some of which exhibited antitumor (Zhou et al., 2016), anti-inflammatory (Hwang et al., 2010), and immunomodulatory activities (Zhao et al., 2001).

Plant Material and Extraction
The fresh stems of D. nobile were collected from Shishan town in May 2018, Hainan, China. After drying, the dried stems (13.0 kg) were shattered. Then, the samples were extracted by 95% ethyl alcohol three times and for 5 days each time. The EtOH extract (716.2 g) was dissolved in H 2 O and extracted three times by petroleum ether (8.0 L), EtOAc (8.0 L), and n-butyl alcohol (8.0 L) successively.

ECD Calculation
The calculations were performed by using the density functional theory (DFT) as carried out in the Gaussian 03 (Frisch et al., 2004). The preliminary conformational distributions search was performed by Sybyl-X 2.0 software. All ground-state geometries were optimized at the B3LYP/6-31G(d) level. Solvent effects of methanol solution were evaluated at the same DFT level by using the SCRF/PCM method (Cammi and Tomasi, 1995). TDDFT at B3LYP/6-31G(d) was employed to calculate the electronic excitation energies and rotational strengths in methanol (Gross et al., 1996).

Bioassay for α-Glycosidase Inhibitory Activity
The method optimized by Jong et al. (2007) was performed in vitro to test the α-glucosidase inhibitory activity of compounds 1-8. Acarbose was used as positive control.

Bioassay for Cytotoxicity
The MTT method optimized by Mosmann (1983) was performed in vitro to test the cytotoxic activity of compounds 1-8. Cisplatin was used as a positive control.

Identification of Compounds 1-4
Compound 1 was obtained as colorless crystals with the molecular formula as C 15 H 21 NO 3 , which was determined based on the pseudo-molecular ion peak at m/z 286.1417 [M + Na] + (calcd. for C 15 H 21 NO 3 Na: 286.1414) in the HRESIMS spectrum. Analysis of its 1D and HSQC NMR spectra (see Supplementary Figures 1, 2, and 4  with one oxygenated, one quaternary carbon (δ C 52.5) and two ester or amide carbonyl groups (δ C 180.3 and δ C 163.7). The above data and sequential COSY correlations (Figure 2 and Supplementary Figure 3 in the Supplementary Material) from H-2 to H-9, as well as from H-4 to H 3 -14 and H 3 -15 through H-13, along with the HMBC correlations (Figure 2 and Supplementary Figure 5 in the Supplementary Material) from H 3 -10 to C-2, C-6 and C-9, from H-2 to C-6 and C-9, from H-3 and H-6 to C-16, and from H-5 to C-1, revealed a picrotoxane-type sesquiterpene skeleton . A detailed comparison of the above data with those of the previously reported dendrobiumane C  disclosed high similarity. The differences between them were that the hydroxymethyl group CH 2 -11 (δ C/H 59.8/4.04, 4.27) and the oxymethine group CH-2 (δ C/H 73.1/3.67) in dendrobiumane C were replaced by a hydrogen atom H-9 (δ H 5.51) and a methine CH-2 (δ C/H 51.2/4.24) linked with a formamide group in 1, respectively, as evidence by COSY correlation from H-9 to H-8, along with the HMBC correlations from H-2 to C-9 and C-11, from H-9 to C-6 and C-10, as well as from H 3 -10 to C-2 and C-6. The ROESY correlations (Figure 3 and Supplementary Figure 6 in the Supplementary Material) from H 3 -10 to H-2, H-6 and H-13, and from H-2 and H-6 to H-13 suggested that H-2, H-6 and H 3 -10 were on the same face of the six-member ring, while H-4 was on the face opposite to them. Due to the complex bridgering lactone skeleton, the chemical structure models analysis of 1 displayed the only possibility of the relative configurations of C-3 and C-5 is that H-3 and H-5 were on the same face of six-member ring with H-2, H-6, and H 3 -10 when the relative configurations of C-1, C-2, C-4 and C-6 were confirmed. To support the above structure elucidation and determine the absolute configuration of 1, a single-crystal X-ray diffraction pattern was obtained using the anomalous scattering of Cu Kα radiation (Figure 4), allowing an explicit assignment of the absolute configuration of 1 as 1S, 2S, 3R, 4S, 5R, and 6S. In order to confirm the absolute configuration assignment, Lactone sector rule (Jexkings et al., 1965) based on ECD data was used. The molecule was viewed from the line on the plane of the ester group along the bisectrix of the O-C=O angle, i.e., as shown in Figure 5 for 4S. The functional group at C-4 lying in the back upper right sector was responsible for the positive CD Cotton effect resulted from n-π * transition of lactone, which was well in accordance with the positive Cotton effect at λ max 223 nm of 1 (Supplementary Figure 24 in Supplementary Material). Hence, compound 1, named as dendroterpene A, was determined to be a picrotoxane-type sesquiterpene.
Compound 2 was also isolated as colorless crystals. The HRESIMS (m/z 302.1376 [M + Na] + , calcd. for C 15 H 21 NO 4 Na: 302.1363) indicated a molecular formula of C 15 H 21 NO 4 . The 1D and 2D NMR data of 2 (Table 1 and Figure 2) were very similar to those of 1, except that an oxygenated quaternary carbon C-6 (δ C 80.6) in 2 replaced signals corresponding to CH-6 in 1 (δ C/H 42.5/2.33). In the HMBC spectrum (Figure 2 and Supplementary Figure 11 in the Supplementary Material) of 2, correlations from H-2, H-4, H-8, H-9, 6-OH, and H 3 -10 to the oxygenated quaternary carbon C-6, together with the molecular formula, suggested that the H-6 in 1 was oxygenated to  Frontiers in Chemistry | www.frontiersin.org a hydroxylated quaternary carbon in 2. The H-2, 6-OH and H 3 -10 were on the same face of the six-member ring, and the H-4 was on the opposite face in 2, as evidence by the ROESY correlations (Figure 3 and Supplementary Figure 12 in the Supplementary Material) of H 3 -10 with H-2, H-13 and 6-OH, and of H-2 and 6-OH with H-13. The relative configurations of C-3 and C-5 in 2 were identical with those of 1 according to the chemical structure models analysis of 2.
The above assignment was further confirmed by a singlecrystal X-ray diffraction pattern obtained using the anomalous scattering of Cu Kα radiation (Figure 4), which also led to an unambiguous assignment of the absolute configuration of 2 as 1R, 2S, 3R, 4S, 5S, and 6R. The absolute configuration of 2 was also confirmed by Lactone sector rule based on ECD data (Jexkings et al., 1965) (Figure 5 and Supplementary Figure 24 in Supplementary Material). Therefore, compound 2 was also identified as a picrotoxane-type sesquiterpene, and named as dendroterpene B.
Compound 3 has the molecular formula of C 15 H 20 O 4 based on HRESIMS (m/z 287.1281 [M + Na] + , calcd for C 15 H 20 O 4 Na:287.1254), suggesting six degrees of unsaturation. 1D NMR and HSQC data displayed three methyls (δ C/H 31.3/1.52, 21.4/1.02 and 20.6/1.04), two methylenes, seven methines including two oxygenated ones (δ C/H 81.5/4.28 and 77.9/4.75), three quaternary carbons including two ester carbonyl carbons (δ C 177.5 and 177.1). The above data of 3 were very similar to those of the previously reported dedrobiumane E . Their structural differences were that two oxygenated quaternary carbons C-6 (δ C 76.8) and C-9 (δ C 83.8) and two oxygenated methine groups CH-7 (δ C/H 65.2/4.05) and CH-8 (δ C/H 60.1/3.86) in dedrobiumane E were replaced by two sp 3 methines (δ C/H 43.6/2.20 and δ C/H 55.2/2.77) and two sp 3 methylenes (δ C/H 32.4/2.15 and δ C/H 27.8/2.01, 2.14) in 3, as proved by the sequential COSY correlations of H-5/H-6/H-7/H-8/H-9, along with the detail interpretation of the HMBC correlations from H-2 to C-6, C-9, and C-11, from H 3 -10 to C-2, C-6, and C-9, from H-3 and H-6 to C-16, from H-5 to C-1, as well as from H-8 to C-11 (Figure 2). The ROESY experiments (Figure 3 and Supplementary Figure 18 in the Supplementary Material) displayed the correlations from H 3 -10 to H-2, H-6, and H-9, as well as the correlations between H-2 and H-13, indicating H-2, H-6, H-9, and H 3 -10 were on the same face, while H-4 was on the face opposite to them. The relative configurations of C-3 and C-5 in 3 were also determined to be consistent with those of 1 and 2 through the same method. The calculated ECD curve for 3 matched well with the experimental one (Figure 6), assigning the absolute configurations of 3 as 1R, 2S, 3R, 4S, 5R, 6S, and 9S (Wang et al., 2014). Thus, the structure of 3 was established as picrotoxane-type sesquiterpene shown in Figure 1, and named as dendroterpene C. Compound 4, colorless crystals, the molecular formula of which was established to be C 15 H 20 O 5 according to the pseudomolecular ion peak at m/z 303.1228 [M + Na] + (calcd. for C 15 H 20 O 5 Na: 303.1203) in the HRESIMS spectrum. The similarity of NMR data (Table 1 and Figure 2) between 3 and 4 indicated that they have the same picrotoxane-type sesquiterpene skeleton. The only structural difference between them was CH-9 (δ C/H 55.2/2.77) in 3 was oxygenated to a hydroxylated quaternary carbon (δ C 85.1) in 4, as supported by sequential COSY correlations of H-2/H-3/H-4/H-5/H-6/H-7/H-8, together with the HMBC correlations from H-2, H-6, H-7, and H 3 -10 to C-9. The stereochemical structure of 4 (1S, 2S, 3R, 4S, 5R, 6S, and 9R), was determined by a single-crystal X-ray diffraction pattern obtained using the anomalous scattering of Cu Kα radiation (Figure 4). From a biosynthetic consideration, the absolute configurations of 4 were deduced be identical to those of 1-3. Hence, compound 4 was also identified as a picrotoxane sesquiterpene as shown in Figure 1, named as dendroterpene D.
The Postulated Biosynthesis Pathway of Compounds 1-4 The carbon skeletons of previously reported picrotoxane-type sesquiterpenes are highly conserved.
The main skeletal changes are that one is the C-11-O-C-2 ring or the C-11-N-C-2 ring closure exemplified by dendrobiumane E and the other is the C-11-O-C-2 ring or the C-11-N-C-2 ring opening illustrated by dedrobiumane B. In our present study, the discovery of 1 and 2 with a new carbon skeleton containing a formamide group, which was derived from unprecedented carbon bond cleavage pattern prompted us to study its plausible biosynthetic pathways (Figure 7). Dendrobiumane B was assumed to be the biosynthetic precursor

Biological Activity
Compounds 1-8 were tested for inhibitory activity against α-glycosidase using the PNPG method and for cytotoxic effects on SGC-7901, K562, A549, BEL-7402, and Hela cell lines using the MTT method (See Supplementary

CONCLUSIONS
In conclusion, four new picrotoxane-type sesquiterpenes (1-4), together with four known compounds (5-8), were isolated from the stems of D. nobile. Compounds 1 and 2 are two new picrotoxane-type sesquiterpenes with a new carbon skeleton containing a formamide group, which may be derived from the previously reported dendrobiumane B skeleton by the C(9)-C(11) carbon bond cleavage. In the previously report, picrotoxane sesquiterpenes exhibited the angiogenesis effect against sunitinibinduced damage (Meng et al., 2017) and inhibitory activity of nerve growth factor mediated neurite outgrowth (Leon et al., 2019). Compound 3 exhibited inhibitory activity against α-glycosidase with IC 50 values of 0.97 mM. However, compound 4 is inactive (IC 50 > 1 mM) against αglycosidase, indicating that the hydroxyl at C-9 may reduce the activity. In addition, the known bibenzyl derivative nobilin E (5) exhibited inhibitory effects on NO production in activated murine macrophage-like cell line RAW 264.7 in the previously report (Zhang et al., 2007b). The bioactivities of the known bibenzyl derivative dendrocandin V (6) have not been reported. In this report, compounds 5 and 6 both exhibited inhibitory activity against α-glycosidase with IC 50 values of 0.03 and 0.68 mM, respectively. In addition, Compound 5 displayed weak cytotoxic effects against SGC-7901, K562, A549, BEL-7402 and Hela cell lines with the IC 50 values of 17. 30, 10.39, 29.03, 20.13, and 22.19 µM, respectively. Compound 6 showed cytotoxic effect against K562 with the IC 50 value of 28.23 µM.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.