neo-Clerodane diterpenoids from Salvia dugesii and their bioactive studies

Salvia dugesii is an invasive plant in Yunnan, China. To tentatively explore its utilization, a systematic phytochemical investigation was carried out on this plant, which led to the isolation of five new neo-clerodane diterpenoids, dugesins C–G (1–5), together with six known ones. Their structures were determined by comprehensive NMR and MS spectroscopic analysis. It was noteworthy that the eleven isolates, composed of five different carbocyclic systems derived from the neo-clerodane diterpenoid skeleton, were reported from the same plant for the first time. The anti-feedantial, cytotoxic, and antiviral activities of the isolates were evaluated. Dugesin F (4) was tested to be a non-toxic antiviral compound against influenza virus FM1. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-011-0016-6 and is accessible for authorized users.


Introduction
The problem caused by invasive species has become increasingly important in recent years. Salvia dugesii Fernald is native to Mexico and was introduced to Yunnan province of China in late 1980s. Its rapid spread in the ecosystem as an invasive plant and has caused increasing threats to the local agricultural and horticultural systems. 1 Clerodane diterpenoids, a type of characteristic secondary metabolites of Salvia species, have been reported frequently with diverse biological activities from different Salvia species distributed in Mexico and Central and South America. [2][3][4][5][6][7] Previously chemical studied on the title plant afforded several neo-clerodane diterpenoids. 8 With the aim of searching for useful metabolites from this invasive plant and continuing our systematic studies of Salvia species, [8][9][10] the chemical constituents of S. dugesii was investigated, and five new neo-clerodane diterpenoids (dugesins C-G, 1-5) were isolated together with six known ones: dugesins A and B, 8 tilifodiolide, 3 isosalvipuberulin, 4 salviandulin E, 5 and salvifaricin. 11,12 In order to assess the potential usage of these isolates, their cytotoxic, antifeedantial, and antiviral activities were evaluated. Dugesin F (4) was shown to be a non-toxic antiviral compound against influenza virus FM1. Reported herein were the isolation, structure, and bioactivity evaluation of these metabolites.

Results and Discussion
*To whom correspondence should be addressed. E-mail: xugang008@mail.kib.ac.cn; xlshen66@hotmail.com The acetone extract of the aerial parts of S. dugesii was repeatedly chromatographed over silica gel, Lichroprep RP-18, and Sephadex LH-20 to afford five new neoclerodane diterpenoids (dugesins C-G, 1-5), and six known ones, dugesins A and B, tilifodiolide, isosalvipuberulin, salviandulin E, and salvifaricin. It was interesting that the eleven isolates, composed of five different carbocyclic systems derived from the normal neo-clerodane diterpenoid skeleton, were reported from the same plant for the first time. It was also noteworthy that the skeleton of dugesins A and D, and salviandulin E, possessing a 6/7 carbocyclic systems derived from the normal neo-clerodane skeleton, respectively, was very rare in nature. 5,6,8,13,14 Dugesin C, possessing a spirocyclic carbocyclic system derived from the normal neo-clerodane skeleton, was the third example of natural metabolites with such a novel carbocyclic skeleton. 15 The major constituent (content about 0.2% in the dry plant sample) of S. dugesii, tilifodiolide, can be seen as the only tetraline-type diterpenoid of clerodanic origin in nature products up to now. 3 In order to assess the potential usage of these isolates, their antifeedantial, cytotoxic, anti-malarial, and antiviral activities were evaluated, and dugesin F (4) was tested to be a non-toxic antiviral compound against influenza virus FM1. It was noteworthy that dugesins C and D, possessing a 6/7 carbocyclic system derived from the normal neo-clerodane skeleton, were two salvigenane diterpenoids which were very rare in nature.
Dugesin C (1) was obtained as white amorphous powder, giving a molecular ion peak at m/z 338 in the EIMS spectrum. It was deduced to have a molecular formula C 20 H 18 O 5 by HRESIMS and the 13 C NMR spectrum ( Table 1). The IR spectrum showed the presence of furan ring (1504 and 874 cm −1 ) and α,β-unsaturated γ-lactone moieties (1754 and 1682cm -1 ). [2][3][4][5][6][7][8] The 13 C NMR and DEPT spectra of 1 exhibited signals for 20 carbons, including two lactonic carbonyls, six quaternary carbons, six methines (including four unsaturated ones and an oxygenated one), and six methylenes (one unsaturated one and one oxygenated). Considering many neo-clerodane diterpenoids have been isolated from different Salvia species previously, along with the spectral features obseverd in the NMR spectra, 1 could be ascribed to be a neo-clerodane diterpenoid. [3][4][5][6][7][8]15 Carefully analysis of the NMR spectral data indicated that the 1D NMR data (Table 1) of 1 are very similar to those of salvileucalin A, a novel spirocyclic diterpenoid with a rearranged neo-clerodane keleton from Salvia leucantha. 15 The differences observed in the 1 H and 13 C NMR spectra were the presence of one methylene (δ C 23.5, C-2) and a methine (δ C 44.6, C-5) signals in 1 instead of the signals for the unsaturated methine at C-2 and the unsaturated quaternary carbon at C-5 in salvileucalin A. These differences suggested that the two double bonds of Δ 2(3) and Δ 4(5) in salvileucalin A was replaced by Δ 3(4) in 1, which can be confirmed by the HMBC correlations from H-20 ( H 5.16 and 5.10, each 1H) to C-1, C-9, C-10, and C-11, from H-3 ( H 6.73, s) to C-1, C-2, C-5, and C-18, and from H-5 ( H 3.16, m) to C-1, C-4, C-6, C-9, C-10, and C-19. In addition, two key correlation systems of H-1/H-2/H-3 and H-5/H-19 in the 1 H-1 H COSY spectrum was evident in 1, which also confirm its major difference between salvileucalin A.
The relative configuration of 1 was elucidated to be the same with that of salvileucalin A by analysis of ROESY spectrum ( Figure 1). The ROESY cross-peaks of H-5/H-6β, H-5/H-1β, H-19/H-20, and H-19/H-6α, established the βorientation of H-5 and C-6. H-12 was deduced to be α-oriented by its NOE correlation with H-20. On the basis of all the above evidence, the structure of 1 was established and named dugesin C.
The HRESIMS indicated that 2 (dugesin D) has a molecular formula of C 20 H 20 O 6 . Spectroscopic data interpretation indicated that the structure of 2 was also closely similar to dugesin A. 8 The differences in the 1D NMR spectral data of 2 compared to dugesin A were the presence of three more methines (including a oxygenated one) instead of two olefinic carbons (C-9 and C-10) and a methylene signal (C-6) in dugesin A, which indicated C-9 and C-10 were two saturated methines and C-6 was substituted by a hydroxyl group. This deduction can be confirmed by HMBC correlations observed from H-6 to C-4, C-5, C-8, and C-10, from H-10 to C-1, C-5, C-9, C-19, and C-20, and from H-9 to C-1, C-5, C-8, C-10, C-11, C-12, and C-20. The relative configuration of 2 was established on the basis of ROESY experiment. The NOE correlations of H-19 with Me-20 and H-6 indicated the α-orientation of both H-6 and Me-20. In addition, the NOE correlations for H-9/H-10 and H-9/H-12 indicated that H-10 and H-12 were both βoriented. So, the structure of 2 can be described as 6βhydroxy-3,8-salvigenadiene, and named dugesin D.   The molecular formula of dugesin E (3) was determined to be C 22 H 24 O 7 from its HRESIMS and NMR spectral data. The 13 C NMR spectrum (Table 1) exhibited signals for seven quaternary carbons, eight methines, five methylenes, and two methyls. A side-by-side comparison of the 1D NMR spectral data of 3 and bacchotricuneatin A showed that 3 was the acetoxyl derivative of bacchotricuneatin A. 16 The acetoxyl group was located at C-6 by the HMBC correlations observed between H-6 ( H 5.33, s) with C-4, C-5, C-8, C-19, and the acetoxyl carbonyl at δ C 169.3 (s). The relative configuration of 3 was suggested to be the same as that of bacchotricuneatin A by the analysis of the ROESY correlations ( Figure 2). The NOE correlation between H-6 and H-19 indicated the α-orientation of H-6. In the same spectrum, the correlations for H-8/H-12, H-8/H-10, Me-20/H-19, and H-1α/H-19 confirmed H-8, H-10, and H-12 were all β-oriented while Me-20 was in the αorientation. Thus, the structure of 3 was elucidated and the trivial name was proposed as dugesin E.
Dugesin F (4) has the molecular formula C 20 H 20 O 6 deduced from the HRESIMS. The NMR spectral data of 4 were closely comparable to those of salvifolin except for the lack of an acetyl group at C-20, 3 which indicated that compound 4 was the 20-deacetyl derivative of salvifolin. This deduction can be confirmed by the HMBC correlations from H-20 to C-8, C-9, C-10, C-11, and C-12. The relative configuration of 4 was indicated to be the same as that of salvifolin by the comparative analysis of the ROESY spectrum ( Figure 3 The molecular formula C 20 H 20 O 6 was determined for dugesin G (5) by the HRESIMS. The 1 H and 13 C NMR signals for one tertiary methyl, a β-substituted furan ring, an carboxyl, and an α,β-unsaturated γ-lactone moieties were evident. Carefully spectroscopic data interpretation indicated that the structure of 5 was very similar to nasimalun A, a neo-clerodane diterpenoid previously isolated from Barringtonia racemosa. 17   It was evident that signals for the methoxyl group in nasimalun A was absent in the 1D NMR spectra of 5. The other notable difference was that the oxygenated methine (C-8) and a methylene (C-7) in nasimalun A were changed into a double bond in 5. This was supported by HMBC correlations from H-7 to C-5, C-6, C-8, C-9, and C-17. In the ROESY spectrum, the correlations of Me-20/H-19, H-19/H-1α, H-1β/H-10 can be found, which indicated Me-20 was in the α-orientation while H-10 was β-oriented. Then, the structure of 5 (dugesin G) was thus established as depicted.
To explore the usage of this invasive plant, metabolites of this plant had been assessed in human tumor cell lines as well as in influenza virus FM1 strain. Firstly, the eleven isolates were evaluated for cytotoxicity toward human tumor cells HepG2, CNE, Hela, and NCI-H460, but none of them exhibited promising antitumor activity with IC 50 < 20 µM. In antiviral assay, sample were tested in vitro anti-influenza ability by Cytopathic effect (CPE) and hemagglutination (HI) test. Under their non-toxic concentrations to dog kidney MDCK cells (non-toxic concentration of 4 to cells is 9.78 uM). dugesin F (4) was the only one exhibiting inhibitory effect on influenza virus FM1 strain causing CPE in MDCK cells. This isolates at 31.25 μg/mL completely inhibited influenza virus FM1 strain in HI test. TC 50 value and IC 50 value of this compound determined by MTT assay were 45.67 μg/mL and 9.43 μg/mL, respectively. Corresponding therapeutic index (TI) was 4.84, implying dugesin F (4) a non-toxic antiviral compound.
Considering the fact that clerodane diterpenoids have reported as a rich source of natural insect antifeedants, 18 the antifeedant activity of the four major constituents tilifodiolide, compound 1, isosalvipuberulin, and salviandulin E was also evaluated. The result indicated that dugesin E and isosalvipuberulin exhibited weak anti-feedant activities with AI (antifeedant index, %) of 6.49 and 14.94, respectively. Tilifodiolide and salviandulin E had no anti-feedant activity at all. Instead, they can improve the Pseudaletia separata Walker to eat fresh leaves of Vicia faba L., especially for tilifodiolide (Table 3).