Dendrowardol C, a novel sesquiterpenoid from Dendrobium wardianum Warner

Dendrowardol C (1)—a novel sesquiterpenoid, with an unprecedented 4/5/6/6 tetracyclic carbon backbone, together with two known cyclopacamphane-type sesquiterpenoids; dendronobilin I (2) and dendrobane A (3) were isolated from the stems of Dendrobium wardianum Warner. The structure of 1 was established on the basis of spectroscopic data and the absolute configuration was determined by single-crystal X-ray diffraction crystallography. The hypothetical biosynthetic pathway of 1 was postulated. Compound 1 showed no cytotoxic activity against human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW480.


Introduction
The stems of several Dendrobium species (Orchidaceae) are used in traditional Chinese medicine mainly for nourishing the stomach, promoting secretion of saliva, and reducing fever. 1 Dendrobium wardianum Warner is an endemic plant distributed mainly in southern Yunnan Province, China and some southeast Asian countries, i.e. Myanmar, Bangladesh, and Thailand. 2 This plant was rarely used as ″Shi-Hu″ ever and previous chemical investigation on this plant has led to the isolation of an picrotoxane-type alkaloid, dendrowardine. 3 Recently, we have isolated three sesquiterpenoids from the stems of D. wardianum Warner 4 and continual chemical investigation of the same collection of this plant led to the isolation of one novel sesquiterpenoid, dendrowardol C (1) with an unprecedented 4/5/6/6 tetracyclic ring system, together with two known cyclopacamphane-type sesquiterpenoids. The new structure was determined on the basis of extensive spectroscopic analysis and the X-ray crystallographic diffraction analysis, while the known sesquiterpenoids were identified as dendronobilin I (2) 5 and dendrobane A (3) 6 by comparison with the literatures. In addition, the hypothetical biosynthetic pathway of 1 was postulated.

Results and Discussion
Compound 1 was isolated as a colorless crystal (MeOH  (Table 1) revealed 15 carbon signals arising from two quaternary carbons (one oxygenated), seven methines (one oxygenated), four methylenes (one oxygenated) and two methyls. Since no C=O and C=C double bonds were dectected according to 13 C NMR and IR data, a tetracyclic structure was required for 1 to fulfill the four degrees of unsaturation. The comparison of 1 H and 13 C NMR spectra data of 1 ( Table 1) with those of dendronobilin I (2) suggested that 1 and 2 were similar in rings B and C, indicating the similar structure of 1 and 2. For the cyclopacamphane-type sesquiterpene in Dendrobium species, C-3 is usually a quaternary carbon without oxygen and C-15 is constantly a methyl or an oxygenated methylene. [5][6][7] Interestingly, an oxygenated quaternary carbon (δ C 78.1) and a methylene carbon (δ C 35.2) signals appeared in 13 C NMR (DEPT) spectrum of compound 1. From the HSQC spectral data of 1, twenty-one protons were assigned unambiguously to thirteen carbons, respectively. The partial structure (rings B and C) *To whom correspondence should be addressed. E-mail: hujiangmiao@mail.kib.ac.cn was constructed by 1 H-1 H COSY correlations of H-1/H-2/H-5/H-6/H-7, H-1/H-7, and H-9/H-10/H-11/H-1, and HMBC correlations from H-9 to C-7 and C-8, from Me-15 to C-7 and C-8. The HMBC ( Figure 1) correlations of H-1, H-2 and H-4 (δ H 2.25, 3.08) with C-3 (δ C 78.1), as well as 1 H-1 H COSY correlation of H-4/H-5, indicated the linkage of C-2/C-3/C-4, which established a four-membered ring C system. Furthermore, the cross-peak of Me-15/C-3 displayed the connecting of C-8/C-3. These data were in good agreement with the spectral data observed for C-3 (an oxygenated quaternary carbon) and C-4 (an methylene carbon). The other correlations in 1 H-1 H COSY spectrum revealed the fragments of C-13/C-12/C-14 and C-11/C-12. Thus, the planar structure of 1 with a novel 4/5/6/6 tetracyclic ring system was proposed as shown in Figure 1.
The correlations of H-1/H-7, H-7/H-11, and H-1/H-6, were observed from the ROESY spectrum ( Figure 1), which revealed that H-1, H-7, H-11 and OH-6 were α-, α-, α-, βoriented, respectively. Since C-3 (in ring A system) was established to be connected to C-8 (in ring C system) in the structure of 1 with a 4/5/6/6 tetracyclic carbon skeleton, Me-15, H-2, and H-5 should be on the same side as H-1 and H-6, possessing α-orientation. Both Me-15 and H-5 were α-oriented, which was also supported by the correlations of H-7/Me-15 and H-5/H-6 in the ROESY spectra. Thus, the relative configurations of all chiral carbons in the molecule except for C-12 were determined as the configuration of the stereocenter at C-12 could not be determined due to free rotation of this center with the C-11-C-12 bond 5 .
The presence of the two structurally related sesquiterpenoids (1 and 2) in the same plant implied that dendrowardol C (1) might be derived from the sesquiterpene cyclosativene. Compound 1 was assayed for its cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) by the MTT assay method, with DDP and taxol as positive controls. The results showed that compound 1 exhibited no significant cytotoxic activity against the above cell lines at the concentration of 40 μM.

Experimental Section
General Experimental Procedures. Melting points were obtained on an X-4 micro melting point apparatus. Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrometer. IR spectra were recorded on a Bruker FT-IR Tensor 27 spectrometer using KBr pellets. 1D and 2D NMR   Extraction and Isolation. The fresh stems of D. wardianum Warner (50 Kg) were extracted with 95% EtOH (24 L × 3) and the ethanol solution was concentrated to a water suspension under reduced pressure by rotary evaporator and then partitioned between CHCl 3 and HCl/H 2 O (pH 2). The aqueous layer was adjusted to pH = 10 with 1 M sodium hydroxide solution and then extracted with CHCl 3 to give an alkaloidal extract (6.0 g). The aqueous phase was then subjected to macroporous resin (D101) chromatography to afford the crude water-soluble material (92.0 g). The water soluble material was subjected to silica gel column chromatography (CC) (CHCl 3 /MeOH, 30:1, 20:1, 15:1, 10:1, 5:1, 0:1) to afford fractions I−VII. Fraction III (8.5 g) was subjected to CC over silica gel (CHCl 3 -MeOH, from 20:1 to 2:1), Sephadex LH-20 chromatography (MeOH), RP-18 CC (MeOH/H 2 O, 1:4−1:0) and further purified through recrystallization from MeOH to yield 1 (5 mg) and dendronobilin I (2) (4 mg). Similarly, fraction VI (8.9 g) was purified through repeated chromatography to afford dendrobane A (3) (2 mg). Cytotoxicity Assay. The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW480. All the cells were cultured in RMPI-1640 or DMEM medium (Hyclone, Logan, UT), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO 2 . Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO). Briefly, 100 μL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 10 5 cells/mL in 100 μL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with DDP and toxal as positive controls. After the incubation, MTT (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 200 μL SDS after the removal of 100 μL of medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC 50 value was calculated by the Reed and Muench's method. 10

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-013-0024-9 and is accessible for authorized users.